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1

Human long-term culture initiating cell assay.  

PubMed

The long-term culture initiating cell (LTC-IC) assay, founded on the bone marrow long-term culture (LTC) system, measures primitive hematopoietic stem cells (termed LTC-IC) based on their capacity to produce myeloid progeny for at least 5 weeks. Adaptations of the LTC system including the use of stromal cell lines, application of limiting dilution analysis, and estimation of average hematopoietic progenitor output per LTC-IC under defined conditions have made it possible to accurately determine LTC-IC content in minimally separated and highly purified cell populations from human hematopoietic tissue sources such as bone marrow, peripheral blood, cord blood, fetal liver as well as cord blood and mobilized peripheral blood. Methodologies for measuring human LTC-IC using bulk cultures, limiting dilution analysis, and single cell cultures are described. PMID:23179836

Liu, Min; Miller, Cindy L; Eaves, Connie J

2013-01-01

2

Defining cell culture conditions to improve human norovirus infectivity assays  

SciTech Connect

Significant difficulties remain for determining whether human noroviruses (hNoV) recovered from water, food, and environmental samples are infectious. Three-dimensional tissue culture of human intestinal cells has shown promise in developing an infectivity assay, but reproducibility, even within a single laboratory, remains problematic. From the literature and our observations, we hypothesized that the common factors that leads to more reproducible hNoV infectivity in vitro requires that the cell line be 1) of human gastrointestinal origin, 2) expresses apical microvilli, and 3) be a positive secretor cell line. The C2BBe1 cell line, which is a brush-border producing clone of Caco-2, meets these three criteria. When challenged with Genogroup II viruses, we observed a 2 Log10 increase in viral RNA titer. A passage experiment with GII viruses showed evidence of the ability to propagate hNoV by both reverse transcription quantitative PCR (qRT-PCR) and microscopy. Using 3-D C2BBe1 cells improves reproducibility of the infectivity assay for hNoV, but the assay can still be variable. Two sources of variability include the cells themselves (mixed phenotypes of small and large intestine) and initial titer measurements using quantitative reverse transcription PCR (qRT-PCR) that measures all RNA vs. plaque assays that measure infectious virus.

Straub, Tim M.; Hutchison, Janine R.; Bartholomew, Rachel A.; Valdez, Catherine O.; Valentine, Nancy B.; Dohnalkova, Alice; Ozanich, Richard M.; Bruckner-Lea, Cindy J.

2013-01-10

3

A cell culture assay for the detection of cardiotoxicity  

SciTech Connect

An important step in minimizing the number of animal experiments in medical research is the study of in vitro model systems. The authors propose the use of shock protein formation, which is a cellular response to cell-damaging stress as an assay to monitor cardiotoxicity. Isolated and cultured cardiac myocytes were prepared by a trypsin digestion method from 18-day-old fetal mice. These cells respond to typical substances inducing shock protein formation in other cellular systems as well as to known cardiotoxins with the de novo synthesis of shock proteins. Pharmaceuticals relevant in transplant medicine were tested for possible cardiotoxic effects: Cyclosporine A evokes shock protein formation at subtherapeutic concentrations. Azathioprine and methyl-prednisolone exert the same effect but at concentration ranges highly above the therapeutic level. The ability to induce shock protein synthesis obviously seems to be restricted to toxic drugs. The data presented demonstrate that the proposed in vitro model system for cardiotoxicity is animal saving and sensitive.

Loew-Friedrich, Iv.; von Bredow, F.; Schoeppe, W. (Department of Nephrology, Hospital of the Johann Wolfgang Goethe-University, Frankfurt am Main (Germany))

1991-04-01

4

A novel microfluidic platform with stable concentration gradient for on chip cell culture and screening assays.  

PubMed

In this work a novel microfluidic platform for cell culture and assay is developed. On the chip a static cell culture region is coupled with dynamic fluidic nutrition supply structures. The cell culture unit has a sandwich structure with liquid channels on the top, the cell culture reservoir in the middle and gas channels on the bottom. Samples can be easily loaded into the reservoir and exchange constantly with the external liquid environment by diffusion. Since the flow direction is perpendicular to the liquid channel on the top of the reservoir, the cells in the reservoir are shielded from shear-force. By assembling the basic units into an array, a steady concentration gradient can be generated. Cell culture models both for continuous perfusion and one-off perfusion were established on the chip. Both adherent and suspended cells were successfully cultured on the chip in 2D and 3D culture modes. After culturing, the trapped cells were recovered for use in a later assay. As a competitive candidate for a standard cell culture and assay platform, this chip is also adaptable for cytotoxicity and cell growth assays. PMID:23884407

Xu, Bi-Yi; Hu, Shan-Wen; Qian, Guang-Sheng; Xu, Jing-Juan; Chen, Hong-Yuan

2013-09-21

5

Heat-Transfer-Method-Based Cell Culture Quality Assay through Cell Detection by Surface Imprinted Polymers.  

PubMed

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. PMID:25654744

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

6

CANDLES, an assay for monitoring GPCR induced cAMP generation in cell cultures.  

PubMed

BackgroundG protein-coupled receptors (GPCRs) represent a physiologically and pharmacologically important family of receptors that upon coupling to G¿S stimulate cAMP production catalyzed by adenylyl cyclase. Thus, developing assays to monitor cAMP production is crucial to screen for ligands in studies of GPCR signaling. Primary cell cultures represent a more robust model than cell lines to study GPCR signaling since they physiologically resemble the parent tissue. Current cAMP assays have two fundamental limitations: 1) absence of cAMP kinetics as competition-based assays require cell lysis and measure only a single time-point, and 2) high variation with separate samples needed to measure consecutive time points. The utility of real-time cAMP biosensors is also limited in primary cell cultures due to their poor transfection efficiency, variable expression levels and inability to select stable clones. We therefore, decided to develop an assay that can measure cAMP not only at a single time-point but the entire cAMP kinetics after GPCR activation in untransfected primary cells.ResultsCANDLES (Cyclic AMP iNdirect Detection by Light Emission from Sensor cells) assay for monitoring cAMP kinetics in cell cultures, particularly in primary cultures was developed. The assay requires co-culturing of primary cells with sensor cells that stably express a luminescent cAMP sensor. Upon GPCR activation in primary cells, cAMP is transferred to sensor cells via gap junction channels, thereby evoking a luminescent read-out. GPCR activation using primary cultures of rat cortical neurons and mouse granulosa cells was measured. Kinetic responses of different agonists to adrenergic receptors were also compared using rat cortical neurons. The assay optimization was done by varying sensor-test cell ratio, using phosphodiesterase inhibitors and testing cell-cell contact requirement.ConclusionsHere we present CANDLES assay based on co-culturing test cells with cAMP-detecting sensor cells. This co-culture setup allows kinetic measurements, eliminates primary cell transfections and reduces variability. A variety of cell types (rat cortical neurons, mouse granulosa cells and established cell lines) and receptors (adrenergic, follicle stimulating hormone and luteinizing hormone/chorionic gonadotropin receptors) were tested for use with CANDLES. The assay is best applied while comparing cAMP generation curves upon different drug treatments to untransfected primary cells. PMID:25366423

Trehan, Ashutosh; Rotgers, Emmi; Coffey, Eleanor T; Huhtaniemi, Ilpo; Rivero-Müller, Adolfo

2014-11-01

7

In Vitro Cell Culture Infectivity Assay for Human Noroviruses  

SciTech Connect

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.

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

8

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

PubMed Central

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

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

2015-01-01

9

Radioimmunofocus assay for quantitation of hepatitis A virus in cell cultures.  

PubMed Central

A new method is described for the quantitation of hepatitis A virus in cell cultures, based on the immune autoradiographic detection of foci of infected cells (radioimmunofoci) developing beneath an agarose overlay 14 days after the inoculation of petri dish cultures of continuous African green monkey kidney cells (BS-C-1). The number of foci developing in each culture was linearly related to the dose of hepatitis A virus (either HM-175 or PA-21 strain) inoculated. Focus development was prevented by prior incubation of virus with specific antisera, and the specificity of the radiolabeled antibody reaction was confirmed in competitive blocking experiments. This new assay method retains many of the advantages of conventional plaque assays for virus. Compared with existing end-dilution methods for the quantitation of hepatitis A virus, the radioimmunofocus assay offers greatly improved accuracy and comparable sensitivity, yet is relatively rapid and highly conservative of reagents. Images PMID:6306048

Lemon, S M; Binn, L N; Marchwicki, R H

1983-01-01

10

Gaussia luciferase-based mycoplasma detection assay in mammalian cell culture.  

PubMed

Mycoplasma contamination in mammalian cell culture is a common problem with serious consequences on experimental data, and yet many laboratories fail to perform regular testing. In this chapter, we describe a simple and sensitive mycoplasma detection assay based on the bioluminescent properties of the Gaussia luciferase reporter. PMID:24166367

Degeling, M Hannah; Bovenberg, M Sarah S; Tannous, Marie; Tannous, Bakhos A

2014-01-01

11

COMPARATIVE TOXICITIES OF DIFFERENT FORMS OF ASBESTOS IN A CELL CULTURE ASSAY  

EPA Science Inventory

Three forms of Union Internationale Contre le Cancer (UICC) asbestos, amosite, crocidolite, and chrysotile, were assayed for their cytotoxicity (inhibition of colony formation) in cell culture. Using embryonic human intestine-derived (I-407) and adult rat liver-derived (ARL-6) ep...

12

A high-throughput, homogeneous microplate assay for agents that kill mammalian tissue culture cells.  

PubMed

Screens for cytostasis/cytoxicity have considerable value for the discovery of therapeutic agents and the investigation of the biology of apoptosis. For instance, genetic screens for proteins, protein fragments, peptides, RNAs, or chemicals that kill tissue culture cells may aid in identifying new cancer therapeutic targets. A microplate assay for cell death is needed to achieve throughputs sufficient to sift through thousands of agents from expression or chemical libraries. The authors describe a homogeneous assay for cell death in tissue culture cells compatible with 96- or 384-well plates. In combination with a previously described system for retroviral packaging and transduction, nearly 6000 expression library clones could be screened per week in a 96-well plate format. The screening system may also prove useful for chemical screens. PMID:12857382

Pierce, Michael; Wang, Chunwei; Rebentisch, Matt; Endo, Mark; Stump, Mark; Kamb, Alexander

2003-06-01

13

A radiolabel-release microwell assay for proteolytic enzymes present in cell culture media  

SciTech Connect

A modified method for the measurement of proteolytic enzyme activity in cell culture-conditioned media has been developed. Using the release of 3H-labeled peptides from 3H-labeled gelatin the method is performed in microwell plates. The substrate is insolubilized and attached to the wells by glutaraldehyde treatment, thus eliminating the need for a precipitation step at the end of the assay. The assay is sensitive, reproducible, and convenient for small sample volumes. The effect of different protease inhibitors on activity can be assessed rapidly allowing an early characterization of the enzyme. It can also be adapted to microplate spectrophotometric analysis by staining residual substrate with Coomassie blue.

Rucklidge, G.J.; Milne, G. (Rowett Research Institute, Bucksburn, Aberdeen (England))

1990-03-01

14

Monitoring regulation of DNA repair activities of cultured cells in-gel using the comet assay  

PubMed Central

Base excision repair (BER) is the predominant cellular mechanism by which human cells repair DNA base damage, sites of base loss, and DNA single strand breaks of various complexity, that are generated in their thousands in every human cell per day as a consequence of cellular metabolism and exogenous agents, including ionizing radiation. Over the last three decades the comet assay has been employed in scientific research to examine the cellular response to these types of DNA damage in cultured cells, therefore revealing the efficiency and capacity of BER. We have recently pioneered new research demonstrating an important role for post-translational modifications (particularly ubiquitylation) in the regulation of cellular levels of BER proteins, and that subtle changes (?20–50%) in protein levels following siRNA knockdown of E3 ubiquitin ligases or deubiquitylation enzymes can manifest in significant changes in DNA repair capacity monitored using the comet assay. For example, we have shown that the E3 ubiquitin ligase Mule, the tumor suppressor protein ARF, and the deubiquitylation enzyme USP47 modulate DNA repair by controlling cellular levels of DNA polymerase ?, and also that polynucleotide kinase phosphatase levels are controlled by ATM-dependant phosphorylation and Cul4A–DDB1–STRAP-dependent ubiquitylation. In these studies we employed a modification of the comet assay whereby cultured cells, following DNA damage treatment, are embedded in agarose and allowed to repair in-gel prior to lysis and electrophoresis. Whilst this method does have its limitations, it avoids the extensive cell culture-based processing associated with the traditional approach using attached cells and also allows for the examination of much more precise DNA repair kinetics. In this review we will describe, using this modified comet assay, our accumulating evidence that ubiquitylation-dependant regulation of BER proteins has important consequences for overall cellular DNA repair capacity. PMID:25076968

Nickson, Catherine M.; Parsons, Jason L.

2014-01-01

15

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay  

PubMed Central

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing ?50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population. PMID:21694689

Armstrong, Philip M.; Andreadis, Theodore G.; Finan, Shannon L.; Shepard, John J.; Thomas, Michael C.

2011-01-01

16

Detection of infectious virus from field-collected mosquitoes by vero cell culture assay.  

PubMed

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing ?50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population. PMID:21694689

Armstrong, Philip M; Andreadis, Theodore G; Finan, Shannon L; Shepard, John J; Thomas, Michael C

2011-01-01

17

Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels  

PubMed Central

This protocol describes a simple but robust microfluidic assay combining three-dimensional (3D) and two-dimensional (2D) cell culture. The microfluidic platform comprises hydrogel incorporating chambers between surface-accessible microchannels. Using this platform, well-defined biochemical and biophysical stimuli can be applied to multiple cell types interacting over distances of <1mm, thereby replicating many aspects of the in vivo microenvironment. Capabilities exist for time-dependent manipulation of flows and concentration gradients as well as high-resolution real-time imaging for observing spatial-temporal single cell behavior, cell-cell communication, cell-matrix interactions and cell population dynamics. These heterotypic cell type assays can be used to study cell survival, proliferation, migration, morphogenesis and differentiation under controlled conditions. Applications include the study of previously unexplored cellular interactions, and have already provided new insights into how biochemical and biophysical factors regulate interactions between populations of different cell types. It takes 3 days to fabricate the system and experiments can run for up to several weeks. PMID:22678430

Shin, Yoojin; Han, Sewoon; Jeon, Jessie S.; Yamamoto, Kyoko; Zervantonakis, Ioannis K.; Sudo, Ryo; Kamm, Roger D.; Chung, Seok

2014-01-01

18

Comparison of In Vitro Cell Culture and a Mouse Assay for Measuring Infectivity of Cryptosporidium parvum  

PubMed Central

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 assays indicated that infectivity and disinfection experiments should be limited to discerning relatively large differences. PMID:12147476

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

2002-01-01

19

Low cost microfluidic cell culture array using normally closed valves for cytotoxicity assay.  

PubMed

A reusable low cost microfluidic cell culture array device (MCCAD) integrated with a six output concentration gradient generator (cGG) and 4×6 arrays of microchamber elements, addressed by a series of row and columnar pneumatically actuated normally closed (NC) microvalves was fabricated for cell-based screening of chemotherapeutic compounds. The poly(dimethylsiloxane) (PDMS) device consists of three layers: fluidic, control and membrane which are held by surface contact and made leak-proof by clamping pressure. The NC valves are actuated by a thick PDMS membrane that was created by a novel method based on the self-assembly of PDMS pre-polymer molecules over a denser calcium chloride solution. The membrane actuated the valves reliably and particulates such as alumina particles (3 µm) and MCF-7 cells (20-24 µm) (2×10(5) cells/mL) were flowed through the valves without causing blockage or leakage and consequently avoiding contamination of the different cell culture elements. The MCCAD was cast and assembled in a standard laboratory without specialist equipment and demonstrated for performing quantitative cell-based cytotoxicity assays of pyocyanine on human breast cancer (MCF-7) cells and assessed for toxic effect on human hepatocyte carcinoma (HepG2) cells as an indicator for liver injury. Then, the MCCAD was demonstrated for sequential drug combinatorial screening involving gradient generation of paclitaxel doses followed by treatment with aspirin doses on the viability of MCF-7 cells. The interaction between paclitaxel and aspirin was evaluated by using the Bliss independence predictive model and results showed reasonable agreement with the model. A robust, portable, easily fabricated and low cost device is therefore shown to conveniently carry out culturing of multiple cell lines for high throughput screening of anti-cancer compounds using minimal reagents. PMID:25127624

Pasirayi, Godfrey; Scott, Simon M; Islam, Meez; O'Hare, Liam; Bateson, Simon; Ali, Zulfiqur

2014-11-01

20

Cell Culture-Taqman PCR Assay for Evaluation of Cryptosporidium parvum Disinfection  

PubMed Central

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 log10 units) with LP-UV (20 mJ/cm2) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with <0.1 log10 unit being inactivated. These results demonstrate the inability of MIOX to inactivate Cryptosporidium. The assay is a valuable tool for the evaluation of disinfection systems for drinking water and recycled water. PMID:12732515

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

2003-01-01

21

Gamma radiation increases endonuclease-dependent L1 retrotransposition in a cultured cell assay.  

PubMed

Long Interspersed Elements (LINE-1s, L1s) are the most active mobile elements in the human genome and account for a significant fraction of its mass. The propagation of L1 in the human genome requires disruption and repair of DNA at the site of integration. As Barbara McClintock first hypothesized, genotoxic stress may contribute to the mobilization of transposable elements, and conversely, element mobility may contribute to genotoxic stress. We tested the ability of genotoxic agents to increase L1 retrotransposition in a cultured cell assay. We observed that cells exposed to gamma radiation exhibited increased levels of L1 retrotransposition. The L1 retrotransposition frequency was proportional to the number of phosphorylated H2AX foci, an indicator of genotoxic stress. To explore the role of the L1 endonuclease in this context, endonuclease-deficient tagged L1 constructs were produced and tested for their activity in irradiated cells. The activity of the endonuclease-deficient L1 was very low in irradiated cells, suggesting that most L1 insertions in irradiated cells still use the L1 endonuclease. Consistent with this interpretation, DNA sequences that flank L1 insertions in irradiated cells harbored target site duplications. These results suggest that increased L1 retrotransposition in irradiated cells is endonuclease dependent. The mobilization of L1 in irradiated cells potentially contributes to genomic instability and could be a driving force for secondary mutations in patients undergoing radiation therapy. PMID:16507671

Farkash, Evan A; Kao, Gary D; Horman, Shane R; Prak, Eline T Luning

2006-01-01

22

Paired image- and FACS-based toxicity assays for high content screening of spheroid-type tumor cell cultures  

PubMed Central

Novel spheroid-type tumor cell cultures directly isolated from patients’ tumors preserve tumor characteristics better than traditionally grown cell lines. However, such cultures are not generally used for high-throughput toxicity drug screens. In addition, the assays that are commonly used to assess drug-induced toxicity in such screens usually measure a proxy for cell viability such as mitochondrial activity or ATP-content per culture well, rather than actual cell death. This generates considerable assay-dependent differences in the measured toxicity values. To address this problem we developed a robust method that documents drug-induced toxicity on a per-cell, rather than on a per-well basis. The method involves automated drug dispensing followed by paired image- and FACS-based analysis of cell death and cell cycle changes. We show that the two methods generate toxicity data in 96-well format which are highly concordant. By contrast, the concordance of these methods with frequently used well-based assays was generally poor. The reported method can be implemented on standard automated microscopes and provides a low-cost approach for accurate and reproducible high-throughput toxicity screens in spheroid type cell cultures. Furthermore, the high versatility of both the imaging and FACS platforms allows straightforward adaptation of the high-throughput experimental setup to include fluorescence-based measurement of additional cell biological parameters.

Trumpi, Kari; Egan, David A.; Vellinga, Thomas T.; Borel Rinkes, Inne H.M.; Kranenburg, Onno

2015-01-01

23

A sensitive, simple assay of mitochondrial ATP synthesis of cultured mammalian cells suitable for high-throughput analysis  

Microsoft Academic Search

A new assay has been developed to measure mitochondrial ATP synthesis of cultured mammalian cells. Cells in a microplate are exposed to streptolysin O to make plasma membranes permeable without damaging mitochondrial function and ATP synthesis is monitored by luciferase. Addition of inhibitors of FoF1-ATP synthase (FoF1), respiratory chain, TCA cycle and ATP\\/ADP translocator, as well as knockdown of ?-subunit

Makoto Fujikawa; Masasuke Yoshida

2010-01-01

24

Validation of a Cell-Free Translation Assay for Detecting Shiga Toxin 2 in Bacterial Culture  

Technology Transfer Automated Retrieval System (TEKTRAN)

We have validated a cell-free translation (CFT) assay for detecting Shiga toxin (Stx). The limit of detection (LOD) for pure Stx2 (PStx2) and partially pure Stx2 (PPStx2) in water reached 20 pg/µl and 3.5 pg/µL respectively without the artificial process of proteolytic activation and reduction of th...

25

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

NASA Technical Reports Server (NTRS)

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.

Ferebee, Robert N.

1992-01-01

26

Polymer-Based Mesh as Supports for Multi-layered 3D Cell Culture and Assays  

PubMed Central

Three-dimensional (3D) culture systems can mimic certain aspects of the cellular microenvironment found in vivo, but generation, analysis and imaging of current model systems for 3D cellular constructs and tissues remain challenging. This work demonstrates a 3D culture system – Cells-in-Gels-in-Mesh (CiGiM) – that uses stacked sheets of polymer-based mesh to support cells embedded in gels to form tissue-like constructs; the stacked sheets can be disassembled by peeling the sheets apart to analyze cultured cells—layer-by-layer—within the construct. The mesh sheets leave openings large enough for light to pass through with minimal scattering, and thus allowing multiple options for analysis—(i) using straightforward analysis by optical light microscopy, (ii) by high-resolution analysis with fluorescence microscopy, or (iii) with a fluorescence gel scanner. The sheets can be patterned into separate zones with paraffin film-based decals, in order to conduct multiple experiments in parallel; the paraffin-based decal films also block lateral diffusion of oxygen effectively. CiGiM simplifies the generation and analysis of 3D culture without compromising throughput, and quality of the data collected: it is especially useful in experiments that require control of oxygen levels, and isolation of adjacent wells in a multi-zone format. PMID:24095253

Simon, Karen A.; Park, Kyeng Min; Mosadegh, Bobak; Subramaniam, Anand Bala; Mazzeo, Aaron; Ngo, Phil M.; Whitesides, George M.

2013-01-01

27

Application of PCR to multiple specimen types for diagnosis of cytomegalovirus infection: comparison with cell culture and shell vial assay.  

PubMed Central

Human cytomegalovirus (CMV) is a herpesvirus that is responsible for significant morbidity and mortality in congenitally infected infants and immunocompromised patients. Antiviral therapies are available, thus making timely diagnosis of significant importance to at-risk patients. A PCR system was devised. The newly devised system, unlike previously described systems, can be applied to a wide variety of specimen types in a clinical microbiology laboratory setting. Specimens from all sites routinely accepted for CMV culture were shown to be acceptable for CMV PCR. Sensitivity and specificity were established in comparison with those of both monolayer culture and shell vial assay (SVA). The sensitivity and specificity of PCR for detection of CMV in specimens exclusive of urine and blood were 97.5 (77 of 79 specimens) and 87.2% (41 of 47 specimens), respectively. The sensitivity and specificity of PCR for urine and blood specimens were 100 (10 of 10) and 95.7% (45 of 47) and 66.7 (4 of 6) and 78.8% (41 of 52), respectively. Discrepancies of positive PCR results with negative culture or SVA results occurred for specimens flanked chronologically by other culture- or SVA-positive specimens and were likely culture failures, increasing the specificity (100%) of PCR. Discrepancies of negative PCR results with positive culture or SVA results occurred in specimens with few cells or infectious foci by SVA or culture and may represent sampling variability associated with low virus titers. PMID:8126204

Miller, M J; Bovey, S; Pado, K; Bruckner, D A; Wagar, E A

1994-01-01

28

Evaluation of a Soluble Tetrazolium\\/Formazan Assay for Cell Growth and Drug Sensitivity in Culture Using Human and Other Tumor Cell Lines1  

Microsoft Academic Search

We have previously described the application of an automated micro- culture tetrazolium assay (MTA) involvingdimethyl sulfoxide solubiliza- tion of cellular-generated 3-{4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- zolium bromide (MTTHormazan to the in vitro assessment of drug effects on cell growth (M. C. Alley et al., Proc. Am. Assen. Cancer Res., 27:389,1986; M. C. Alley et al.. Cancer Res. 48:589-601,1988). There are several inherent disadvantages of

Dominic A. Scudiere; Robert H. Shoemaker; Kenneth D. Paul; Anne Monks; Siobhan Tierney; Thomas H. Nofziger; Michael J. Currens; Donna Seniff; Michael R. Boyd

29

Cytotoxicity of mycotoxins evaluated by the MTT-cell culture assay  

Microsoft Academic Search

The application of a modified colorimetric bioassay for the evaluation of the biological effects of mycotoxins is reported. Using three different monolayer cell lines (swine kidney, Madin Darby canine kidney, HeLa) the influence of nine different mycotoxins on the cellular methylthiazoltetrazolium (MTT)-cleavage activity was evaluated. The yellow tetrazolium salt MTT is converted by mitochondrial dehydrogenases of metabolically active cells to

Monika Hanelt; Manfred Gareis; Birgit Kollarczik

1994-01-01

30

Quantum dot-based assay for Cu(2+) quantification in bacterial cell culture.  

PubMed

A simple and sensitive method for quantification of nanomolar copper with a detection limit of 1.2×10(-10)M and a linear range from 10(-9) to 10(-8)M is reported. For the most useful analytical concentration of quantum dots, 1160?g/ml, a 1/Ksv value of 11?M Cu(2+) was determined. The method is based on the interaction of Cu(2+) with glutathione-capped CdTe quantum dots (CdTe-GSH QDs) synthesized by a simple and economic biomimetic method. Green CdTe-GSH QDs displayed the best performance in copper quantification when QDs of different sizes/colors were tested. Cu(2+) quantification is highly selective given that no significant interference of QDs with 19 ions was observed. No significant effects on Cu(2+) quantification were determined when different reaction matrices such as distilled water, tap water, and different bacterial growth media were tested. The method was used to determine copper uptake kinetics on Escherichia coli cultures. QD-based quantification of copper on bacterial supernatants was compared with atomic absorption spectroscopy as a means of confirming the accuracy of the reported method. The mechanism of Cu(2+)-mediated QD fluorescence quenching was associated with nanoparticle decomposition. PMID:24433980

Durán-Toro, V; Gran-Scheuch, A; Órdenes-Aenishanslins, N; Monrás, J P; Saona, L A; Venegas, F A; Chasteen, T G; Bravo, D; Pérez-Donoso, J M

2014-04-01

31

Basics of Cell Culture  

NSDL National Science Digital Library

These manuals are used in the Stem Cell Culture Course at City College of San Francisco. This course is about general mammalian cell culture techniques but includes a laboratory exercise using stem cells (takes 3 weeks to complete). The course is taught to high school students but the materials are also used for college students. Laboratory exercises provide instruction in basic techniques of routine cell culture using common cell lines before progressing to differentiation of mouse embryonic stem cells. Photographs and explanations of common equipment (laminar flow hood, inverted microscope, etc.) and reagents are provided. Laboratory exercises include the following: Basic Aseptic Technique; Media Preparation; Plating cells from frozen stock; Cell counting and plating; Survival assay (UV); Live Cell Identification; Transfection; Freezing cells; Stem cell differentiation. A student lab manual and an instructor manual are provided.

Afshar, Golnar

32

Fatty acid hydroxytyrosyl esters: structure/antioxidant activity relationship by ABTS and in cell-culture DCF assays.  

PubMed

A large series of hydroxytyrosyl esters of C2-C18 fatty acids with increasing lipophilicity was prepared by a new highly efficient method based on acylation of methylorthoformate-protected hydroxytyrosol. All products were tested for relative antioxidant effect using ABTS assays in ethanolic medium and DCF assays in L6 cells. No linear correlation between lipophilicity and antioxidant effect was found. ABTS assays showed a growing antioxidant capacity, with respect to hydroxytyrosol, only for medium-sized ester chains (C4-C10) and a nearly constant capacity for the higher homologues. This has been rationalized by molecular dynamics experiments in terms of partial shielding of the catecholic hydroxyls by long-chain esters. A similar and dose-dependent pattern was observed in DCF assays in L6 cells, but a sharp antioxidant activity drop resulted for long-chain esters, probably due to membrane entrapment. PMID:20397651

Tofani, Daniela; Balducci, Valentina; Gasperi, Tecla; Incerpi, Sandra; Gambacorta, Augusto

2010-05-12

33

Microfluidic Cell Culture and Its Application in High Throughput Drug Screening: Cardiotoxicity Assay for hERG Channels  

E-print Network

Microfluidic Cell Culture and Its Application in High Throughput Drug Screening: Cardiotoxicity, WI, USA, 53706 Abstract Evaluation of drug cardiotoxicity is essential to the safe development identifying lead compounds from drug screens is to measure their cardiotoxicity. Numerous lead compounds

Beebe, David J.

34

An improved infectivity assay combining cell culture with real-time PCR for rapid quantification of human adenoviruses 41 and semi-quantification of human adenovirus in sewage.  

PubMed

A protocol for the rapid detection and semi-quantification of human enteric adenovirus based on the quantification of viral mRNA during cell culture infectivity assay was developed. Infectivity assays for adenovirus incorporated cell culture and reverse transcription real-time PCR, where viral mRNA detection was used to monitor the progress of adenovirus infection (CC/mRNA qPCR). The cell line used was G293. This specific infectivity assay was calibrated against different initial concentrations of human adenovirus 41. In addition, the expression of the host's housekeeping (HK) gene, GAPDH, served as internal control for the mRNA assays for quality assurance of the mRNA extraction and reverse transcription steps. The concentrations of infectious human adenoviruses in different sewage samples were estimated semi-quantitatively using the CC/mRNA qPCR assay and calibration obtained for adenovirus 41. A linear relationship between concentrations of viral mRNA (hexon gene) and infectious units was observed between 10(7) to 10(1) infectious units per assay (R(2) = 0.97) in samples analyzed 3-5 days post infection. The expressions of host cell GAPDH gene were not significantly affected by infections with different concentrations of human adenovirus 41, and between virus positive and negative cell cultures (p > 0.1). The estimated concentrations of human adenoviruses in sewage samples ranged between 10(2) to 10(3) mRNA-IU/L. Most of the viruses detected in sewage samples were from human adenovirus species F. The CC/mRNA qPCR assay can be used for quantifying infectious human adenovirus 41, estimating the levels of human adenoviruses in sewage samples, and applied to other sample settings. The CC/mRNA qPCR protocol described here represents an improvement in the detection of human enteric adenoviruses by reducing incubation time (5 days); whereas the conventional cell culture method requires longer incubation periods (10-20 days). More importantly, this protocol can be used to more rapidly and semi-quantitatively estimate the levels of infectious human adenoviruses in environmental samples. PMID:23579085

Rodríguez, Roberto A; Polston, Patsy M; Wu, Ming Jing; Wu, Jianyong; Sobsey, Mark D

2013-06-01

35

Stable isotope labeling by amino acids in cell culture-based liquid chromatography-mass spectrometry assay to measure microtubule dynamics in neuronal cell cultures.  

PubMed

Microtubules (MTs) are highly dynamic polymers composed of ?- and ?-tubulin heterodimers. Dysregulation of MT dynamics in neurons may be a contributing factor in the progression of various neurodegenerative diseases. We developed a stable isotope labeling by amino acids in cell culture (SILAC)-based liquid chromatography-mass spectrometry (LC-MS) method to measure the fraction of [(13)C6]leucine-labeled ?-tubulin-derived surrogate peptides. Using this approach, we measured the time course of incorporation of [(13)C6]leucine label into the MT and dimer pools isolated from cycling cells and rat primary hippocampal neurons. We found that the MT pool is in rapid equilibrium with the dimer pool in the cycling cells, consistent with rapid MT polymerization/depolymerization during cell proliferation. Conversely, in neurons, we found that labeling of the MT pool was rapid, whereas the dimer pool was delayed. These results suggest that newly synthesized ?-tubulin is first incorporated into MTs or complexes that co-sediment with MTs and that appearance of labeled ?-tubulin in the dimer pool may be a consequence of MT depolymerization or breakdown. Our results demonstrate that a SILAC-based approach can be used to measure MT dynamics and may have utility for exploring MT dysregulation in various models of neurodegenerative disease. PMID:25175011

Polson, Craig; Cantone, Joseph L; Wei, Cong; Drexler, Dieter M; Meredith, Jere E

2014-12-01

36

Development of a combined in vitro cell culture--quantitative PCR assay for evaluating the disinfection performance of pulsed light for treating the waterborne enteroparasite Giardia lamblia.  

PubMed

Giardia lamblia is a flagellated protozoan parasite that is recognised as a frequent cause of water-borne disease in humans and animals. We report for the first time on the use of a combined in vitro HCT-8 cell culture-quantitative PCR assay for evaluating the efficacy of using pulsed UV light for treating G. lamblia parasites. Findings showed that current methods that are limited to using vital stains before and after cyst excystation are not appropriate for monitoring or evaluating cyst destruction post PUV-treatments. Use of the human ileocecal HCT-8 cell line was superior to that of the human colon Caco-2 cell line for in vitro culture and determining PUV sensitivity of treated cysts. G. lamblia cysts were also shown to be more resistant to PUV irradiation compared to treating similar numbers of Cryptosporidium parvum oocysts. These observations also show that the use of this HCT-8 cell culture assay may replace use of animal models for determining disinfection performances of PUV for treating both C. parvum and G. lamblia. PMID:24929148

Garvey, Mary; Stocca, Alessia; Rowan, Neil

2014-09-01

37

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.  

PubMed

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. PMID:25658121

Gisder, Sebastian; Genersch, Elke

2015-01-01

38

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  

PubMed Central

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. PMID:25658121

Gisder, Sebastian; Genersch, Elke

2015-01-01

39

Transducin-alpha C-terminal mutations prevent activation by rhodopsin: a new assay using recombinant proteins expressed in cultured cells.  

PubMed Central

We have measured the activation by recombinant rhodopsin of the alpha-subunit (alpha 1) of retinal transducin (Gt, also recombinant) using a new assay. Cultured cells are transiently transfected with DNAs encoding opsin and the three subunits of Gt (alpha t, beta 1 and gamma 1). In the microsomes of these cells, incubated with 11-cis-retinal, light causes the rapid activation of Gt, as measured by the ability of GTP gamma S to protect alpha t fragments from proteolytic degradation. The activation of Gt is also observed when all-trans-retinal is added to microsomes under constant illumination. Activation depends on both opsin and retinal. Opsin mutants with known defects in activating Gt show similar defects in this assay. alpha t mutations that mimic the corresponding mutations in the alpha-subunit of Gs also produce qualitatively similar effects in this assay. As a first step in a strategy aimed at exploring the relationships between structure and function in the interactions of receptors with G proteins, we tested mutant alpha t proteins with alanine substituted for each of the 10 amino acids at the C-terminus, a region known to be crucial for interactions with rhodopsin. Alanine substitution at four positions moderately (K341) or severely (L344, G348, L349) impairs the susceptibility of alpha 1 to activation by rhodopsin. All four mutants retain their ability to be activated by AIF-4. Two other substitutions (N343 and F350) resulted in very mild defects, while substitutions at the remaining four positions (E342, K345, D346 and C347) had no effect. In combination with previous observations, these results constrain models of the interaction of the C-terminus of alpha t with rhodopsin. Images PMID:7556089

Garcia, P D; Onrust, R; Bell, S M; Sakmar, T P; Bourne, H R

1995-01-01

40

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

PubMed

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

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

2012-04-01

41

A Voltage-Sensitive Dye-Based Assay for the Identification of Differentiated Neurons Derived from Embryonic Neural Stem Cell Cultures  

Microsoft Academic Search

BackgroundPluripotent and multipotent stem cells hold great therapeutical promise for the replacement of degenerated tissue in neurological diseases. To fulfill that promise we have to understand the mechanisms underlying the differentiation of multipotent cells into specific types of neurons. Embryonic stem cell (ESC) and embryonic neural stem cell (NSC) cultures provide a valuable tool to study the processes of neural

Richardson N. Leão; Amilcar Reis; Amanda Emirandetti; Michalina Lewicka; Ola Hermanson; André Fisahn; Wei-Chun Chin

2010-01-01

42

A sensitive and specific ES-31 antigen detection based fluorometric assay for confirmation of Mycobacterium tuberculosis in cell culture.  

PubMed

Confirmation of presence of M. tuberculosis bacilli on microscopic examination is very important in diagnosis of tuberculosis. The present study was undertaken to find the usefulness of mycobacterial ES-31 serine protease as a marker to detect tuberculosis bacilli using fluorescein isothiocyanate conjugated anti-ES-31 serine protease antibody. This immunofluorescence method was compared with Ziehl-Neelsen and auramine-O staining methods for detection of tuberculosis bacilli. Slides were prepared for each serially diluted tuberculosis H37Ra bacilli (1 x 10(7) bacilli/ml to 5 bacilli/ml). Slides for each dilution group were stained by ZN method, auramine-O and immunostaining methods using fluorescein isothiocyanate conjugated anti-ES-31 serine protease antibody. ZN staining method showed efficacy for detection of M. tuberculosis H37Ra upto 1 x 10(4) bacilli/ml while auramine-O method showed upto 1 x 10(2) bacilli/ml. The presence of bacilli was indicated by green fluorescence on immunostaining using anti-ES-31 antibody conjugate and this method was effective upto 10 bacilli/ml. The slides which were negative for ZN (1 x 10(3) cells/ml) and auramine-O (100 cells/ml) method showed positivity on restaining with immunofluorescent staining method. The results of this preliminary study showed that immunofluorescent staining method using specific anti-ES-31 antibody conjugate was more sensitive for detection of tuberculosis bacilli than ZN and auramine-O methods in samples of laboratory strain. The utility of this method will be studied further in clinical specimens. PMID:21614896

Majumdar, Anindita; Wankhade, Gauri; Kamble, Pranita D; Joshi, Deepti; Harinath, B C

2011-04-01

43

Histatins are the major wound-closure stimulating factors in human saliva as identified in a cell culture assay.  

PubMed

Wounds in the oral cavity heal much faster than skin lesions. Among other factors, saliva is generally assumed to be of relevance to this feature. Rodent saliva contains large amounts of growth factors such as epidermal growth factor (EGF) and nerve growth factor (NGF). In humans, however, the identity of the involved compounds has remained elusive, especially since EGF and NGF concentrations are approximately 100,000 times lower than those in rodent saliva. Using an in vitro model for wound closure, we examined the properties of human saliva and the fractions that were obtained from saliva by high-performance liquid chromotography (HPLC) separation. We identified histatin 1 (Hst1) and histatin 2 (Hst2) as major wound-closing factors in human saliva. In contrast, the d-enantiomer of Hst2 did not induce wound closure, indicating stereospecific activation. Furthermore, histatins were actively internalized by epithelial cells and specifically used the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway, thereby enhancing epithelial migration. This study demonstrates that members of the histatin family, which up to now were implicated in the antifungal weaponry of saliva, exert a novel function that likely is relevant for oral wound healing. PMID:18650243

Oudhoff, Menno J; Bolscher, Jan G M; Nazmi, Kamran; Kalay, Hakan; van 't Hof, Wim; Amerongen, Arie V Nieuw; Veerman, Enno C I

2008-11-01

44

Polyalkoxybenzenes from plants. 5. Parsley seed extract in synthesis of azapodophyllotoxins featuring strong tubulin destabilizing activity in the sea urchin embryo and cell culture assays.  

PubMed

A series of 4-azapodophyllotoxin derivatives with modified rings B and E have been synthesized using allylpolyalkoxybenzenes from parsley seed oil. The targeted molecules were evaluated in vivo in a phenotypic sea urchin embryo assay for antimitotic and tubulin destabilizing activity. The most active compounds identified by the in vivo sea urchin embryo assay featured myristicin-derived ring E. These molecules were determined to be more potent than podophyllotoxin. Cytotoxic effects of selected molecules were further confirmed and evaluated by conventional assays with A549 and Jurkat human leukemic T-cell lines including cell growth inhibition, cell cycle arrest, cellular microtubule disruption, and induction of apoptosis. The ring B modification yielded 6-OMe substituted molecule as the most active compound. Finally, in Jurkat cells, compound induced caspase-dependent apoptosis mediated by the apical caspases-2 and -9 and not caspase-8, implying the involvement of the intrinsic caspase-9-dependent apoptotic pathway. PMID:21916509

Semenova, Marina N; Kiselyov, Alex S; Tsyganov, Dmitry V; Konyushkin, Leonid D; Firgang, Sergei I; Semenov, Roman V; Malyshev, Oleg R; Raihstat, Mikhail M; Fuchs, Fabian; Stielow, Anne; Lantow, Margareta; Philchenkov, Alex A; Zavelevich, Michael P; Zefirov, Nikolay S; Kuznetsov, Sergei A; Semenov, Victor V

2011-10-27

45

An Enzyme-Linked Immunosorbent Assay for the Rapid Quantification of Intracellular and Extracellular Guanosine 3?,5?-cyclic Monophosphate in Cultured Glial Cells  

Microsoft Academic Search

A sensitive enzyme-linked, indirect immunosorbent assay (ELISA) for the determination of guanosine 3',5'-cyclic monophosphate (cGMP) in glial cells is described. The assay uses an antiserum produced against succinylated cGMP and is based on the competition between endogenous cGMP and a fixed amount of immobilized antigen. The antibody exhibited a high degree of specificity with negligible cross reactivity with other nucleotides

John Wellard; Brigitte Blind; Bernd Hamprecht

2004-01-01

46

Mutation assays involving blood cells that metabolize toxic substances  

DOEpatents

A line of human blood cells which have high levels of oxidative activity (such as oxygenase, oxidase, peroxidase, and hydroxylase activity) is disclosed. Such cells grow in suspension culture, and are useful to determine the mutagenicity of xenobiotic substances that are metabolized into toxic or mutagenic substances. Mutation assays using these cells, and other cells with similar characteristics, are also disclosed.

Crespi, Charles L. (Downers Grove, IL); Thilly, William G. (Winchester, MA)

1985-01-01

47

A Voltage-Sensitive Dye-Based Assay for the Identification of Differentiated Neurons Derived from Embryonic Neural Stem Cell Cultures  

PubMed Central

Background Pluripotent and multipotent stem cells hold great therapeutical promise for the replacement of degenerated tissue in neurological diseases. To fulfill that promise we have to understand the mechanisms underlying the differentiation of multipotent cells into specific types of neurons. Embryonic stem cell (ESC) and embryonic neural stem cell (NSC) cultures provide a valuable tool to study the processes of neural differentiation, which can be assessed using immunohistochemistry, gene expression, Ca2+-imaging or electrophysiology. However, indirect methods such as protein and gene analysis cannot provide direct evidence of neuronal functionality. In contrast, direct methods such as electrophysiological techniques are well suited to produce direct evidence of neural functionality but are limited to the study of a few cells on a culture plate. Methodology/Principal Findings In this study we describe a novel method for the detection of action potential-capable neurons differentiated from embryonic NSC cultures using fast voltage-sensitive dyes (VSD). We found that the use of extracellularly applied VSD resulted in a more detailed labeling of cellular processes compared to calcium indicators. In addition, VSD changes in fluorescence translated precisely to action potential kinetics as assessed by the injection of simulated slow and fast sodium currents using the dynamic clamp technique. We further demonstrate the use of a finite element model of the NSC culture cover slip for optimizing electrical stimulation parameters. Conclusions/Significance Our method allows for a repeatable fast and accurate stimulation of neurons derived from stem cell cultures to assess their differentiation state, which is capable of monitoring large amounts of cells without harming the overall culture. PMID:21079795

Emirandetti, Amanda; Lewicka, Michalina; Hermanson, Ola; Fisahn, André

2010-01-01

48

Fifty Percent Tissue Culture Infective Dose Assay for Determining the Titer of Infectious Human Herpesvirus 8  

PubMed Central

We have developed a human herpesvirus 8 (HHV-8) 50% tissue culture infective dose (TCID50) assay using the T1H6-DC-SIGN cell line. Infection of T1H6-DC-SIGN cells with HHV-8 induces expression of ?-galactosidase, which was used to determine TCID50 levels. Validation of TCID50 values was performed by immunofluorescence assay of HHV-8 infection of immature dendritic cells at various TCID50 doses. PMID:23554189

Nadgir, Sagar V.; Hensler, Heather R.; Knowlton, Emilee R.; Rinaldo, Charles R.; Rappocciolo, Giovanna

2013-01-01

49

Mammalian Cell Culture  

NSDL National Science Digital Library

This "Course-in-a-Box" from Bio-Link is a good starting point for instructors to develop a course on how to maintain mammalian cells in culture. Students will learn "basic techniques of routine cell culture using common cell lines before progressing to differentiation of mouse embryonic stem cells." Laboratories include Basic Aseptic Technique, Media Preparation, and Plating Cells from Frozen Stock. Materials include an Instructor Laboratory Manual, Student Laboratory Manual, Problem Sets, and Quizzes. A free login is required to access the materials.

50

Comparison of a New Gold Immunochromatographic Assay for the Rapid Diagnosis of the Novel Influenza A (H7N9) Virus with Cell Culture and a Real-Time Reverse-Transcription PCR Assay  

PubMed Central

We assessed a colloidal gold immunochromatographic assay (GICA) for rapid detection of influenza A (H7N9) and compared it with reverse-transcription-polymerase chain reaction (RT-PCR) and viral culture. Samples from 35 H7N9 infected patients were collected, including 45 throat swab samples, 56 sputum samples, and 39 feces samples. All samples were tested by GICA, viral culture, and RT-PCR. GICA specifically reacted with recombinant HA proteins, virus lysates, and clinical samples from H7 subtype viruses. Compared with RT-PCR, GICA demonstrated low sensitivity (33.33%) but high specificity (97.56%). The positive rate of GICA tests for samples collected in the period from 8 to 21 days after contact with poultry was much higher than those for samples collected before or after this period. Compared with viral culture, GICA showed sensitivity of 91.67% and specificity of 82.03%. Sputum specimens were more likely to test positive for H7N9 virus than samples from throat swabs and feces. The GICA-based H7 test is a reliable, rapid, and convenient method for the screening and diagnosis of influenza A (H7N9) disease, especially for the sputum specimens with high viral load. It may be helpful in managing H7N9 epidemics and preliminary diagnosis in early stages in resource-limited settings. PMID:24822207

Wu, Nanping; Peng, Xiaorong; Yao, Hangping; Lu, Xiangyun; Chen, Yu; Wu, Haibo; Xie, Tiansheng; Cheng, Linfang; Liu, Fumin; Kang, Keren; Tang, Shixing; Li, Lanjuan

2014-01-01

51

Comparison of a new gold immunochromatographic assay for the rapid diagnosis of the novel influenza A (H7N9) virus with cell culture and a real-time reverse-transcription PCR assay.  

PubMed

We assessed a colloidal gold immunochromatographic assay (GICA) for rapid detection of influenza A (H7N9) and compared it with reverse-transcription-polymerase chain reaction (RT-PCR) and viral culture. Samples from 35 H7N9 infected patients were collected, including 45 throat swab samples, 56 sputum samples, and 39 feces samples. All samples were tested by GICA, viral culture, and RT-PCR. GICA specifically reacted with recombinant HA proteins, virus lysates, and clinical samples from H7 subtype viruses. Compared with RT-PCR, GICA demonstrated low sensitivity (33.33%) but high specificity (97.56%). The positive rate of GICA tests for samples collected in the period from 8 to 21 days after contact with poultry was much higher than those for samples collected before or after this period. Compared with viral culture, GICA showed sensitivity of 91.67% and specificity of 82.03%. Sputum specimens were more likely to test positive for H7N9 virus than samples from throat swabs and feces. The GICA-based H7 test is a reliable, rapid, and convenient method for the screening and diagnosis of influenza A (H7N9) disease, especially for the sputum specimens with high viral load. It may be helpful in managing H7N9 epidemics and preliminary diagnosis in early stages in resource-limited settings. PMID:24822207

Jin, Changzhong; Wu, Nanping; Peng, Xiaorong; Yao, Hangping; Lu, Xiangyun; Chen, Yu; Wu, Haibo; Xie, Tiansheng; Cheng, Linfang; Liu, Fumin; Kang, Keren; Tang, Shixing; Li, Lanjuan

2014-01-01

52

Mammalian Cell Culture Simplified.  

ERIC Educational Resources Information Center

A tissue culture experiment that does not require elaborate equipment and that can be used to teach sterile technique, the principles of animal cell line maintenance, and the concept of cell growth curves is described. The differences between cancerous and normal cells can be highlighted. The procedure is included. (KR)

Moss, Robert; Solomon, Sondra

1991-01-01

53

Mutation assays involving blood cells that metabolize toxic substances  

DOEpatents

The present invention pertains to a line of human blood cells which have high levels of oxidative activity (such as oxygenase, oxidase, peroxidase, and hydroxylase activity). Such cells grow in suspension culture, and are useful to determine the mutagenicity of xenobiotic substances that are metabolized into toxic or mutagenic substances. The invention also includes mutation assays using these cells, and other cells with similar characteristics.

Crespi, Charles L. (Marblehead, MA); Thilly, William G. (Winchester, MA)

1999-01-01

54

Mutation assays involving blood cells that metabolize toxic substances  

DOEpatents

The present invention pertains to a line of human blood cells which have high levels of oxidative activity (such as oxygenase, oxidase, peroxidase, and hydroxylase activity). Such cells grow in suspension culture, and are useful to determine the mutagenicity of xenobiotic substances that are metabolized into toxic or mutagenic substances. The invention also includes mutation assays using these cells, and other cells with similar characteristics. 3 figs.

Crespi, C.L.; Thilly, W.G.

1999-08-10

55

Bone marrow stromal cell assays – in vitro and in vivo  

PubMed Central

Summary Populations of bone marrow stromal cells (BMSCs, also known as bone marrow-derived “mesenchymal stem cells”) contain a a subset of cells that are able to recapitulate the formation of a bone/marrow organ (skeletal stem cells, SSCs). The biological properties of BMSC cultures are assessed by a variety of assays, both in vitro and in vivo. Application of these assays in an appropriate fashion provide a great deal of information on the role of BMSCs, and the subset of SSCs, in health and in disease. PMID:24482181

Robey, Pamela Gehron; Kuznetsov, Sergei A.; Riminucci, Mara; Bianco, Paolo

2014-01-01

56

Cell-culture bioreactors  

SciTech Connect

When contrasted with microbial fermentation, the characteristics having a bearing on the design and operation of cell-culture bioreactors are fragility, steam sensitivity and anchorage requirements of cells, heat lability and foaming of proteins and other components of cell culture media. Design details of agitation and gas supply, bearings, seals and drives, foam control and sterilization, temperature, oxygen and pH control, water, air and gas purification, liquid feeding and level control, gas exhaust analysis and disposal, handling of liquid effluent and bioreactor installation and scale up are given.

Beck, C.; Stiefel, H.; Stinnett, T.

1987-02-16

57

A root hair assay to expedite cell death research.  

PubMed

Programmed cell death can be defined as an organized cellular destruction and can be activated throughout plant development, as a defense response against invading pathogens or during environmental stress. The root hair assay presented herein enables in vivo quantitative measurements of programmed cell death based on the morphological changes of dying root hairs. Application of this novel, simple technique eliminates the need for establishing cell suspension cultures, resulting in a significant reduction in time, cost, and labor input. Here, we present a detailed root hair assay protocol for the dicotyledonous model plant Arabidopsis thaliana, where results from germination to scoring of cell death can be obtained within 7 days. We also suggest and present a panel of cell death inducing treatments which can be used to study abiotic stress- and mycotoxin-induced programmed cell death in the root hair system in Arabidopsis. A root hair assay protocol for the monocotyledonous model species Brachypodium distachyon is also included. PMID:25408445

Kacprzyk, Joanna; McCabe, Paul F

2015-01-01

58

Optimization of NRU assay in primary cultures of Eisenia fetida for metal toxicity assessment.  

PubMed

Coelomocytes, immunocompetent cells of lumbricids, have received special attention for ecotoxicological studies due to their sensibility to pollutants. Their in vitro responses are commonly quantified after in vivo exposure to real or spiked soils. Alternatively, quantifications of in vitro responses after in vitro exposure are being studied. Within this framework, the present study aimed at optimizing the neutral red uptake (NRU) assay in primary culture of Eisenia fetida coelomocytes for its application in soil toxicity testing. Optimized assay conditions were: earthworm depuration for 24 h before retrieving coelomocytes by electric extrusion; 2 × 10(5) seeded cells/well (200 µl) for the NRU assay and incubation for 1 h with neutral red dye. Supplementation of the culture medium with serum was not compatible with the NRU assay, but coelomocytes could be maintained with high viability for 3 days in a serum-free medium without replenishment. Thus, primary cultures were used for 24 h in vitro toxicity testing after exposure to different concentrations of Cd, Cu, Ni and Pb (ranging from 0.1 to 100 ?g/ml). Primary cultures were sensitive to metals, the viability declining in a dose-dependent manner. The toxicity rank was, from high to low, Pb > Ni > Cd > Cu. Therefore, it can be concluded that the NRU assay in coelomocytes in primary cultures provides a sensitive and prompt response after in vitro exposure to metals. PMID:25011921

Irizar, Amaia; Duarte, Daniel; Guilhermino, Lucia; Marigómez, Ionan; Soto, Manu

2014-09-01

59

Molluscan cells in culture: primary cell cultures and cell lines  

PubMed Central

In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome. PMID:24198436

Yoshino, T. P.; Bickham, U.; Bayne, C. J.

2013-01-01

60

Comparison of fortified calf serum, serum substitutes and fetal calf serum with or without extenders for propagation of cell cultures for virus plaque assays.  

PubMed

Two studies comparing sewage-isolated and laboratory stock viruses were conducted to determine if alternative forms of serum or serum extenders could be used in place of fetal bovine serum without a significant loss of viral titer. In the first study, BGM cells were grown in standard MEM-L15 medium which was supplemented with Nuserum, Sigma serum replacement (CPSR-1), HyClone defined iron supplemented calf bovine serum, fetal bovine serum (FBS) or FBS supplemented with either SerXtend or Mito serum extenders. Comparison of virus titers showed that CPSR-1 gave the best overall results and was comparable to FBS. Of the serum extenders, only SerXtend improved virus recovery from sewage samples. In the second study, all sera were tested with and without SerXtend. In these experiments, SerXtend enhanced virus sensitivity of the BGM cell line grown in the HyClone serum but reduced the sensitivity of those cultured in Sigma serum. In both series, the growth of BGM cells was monitored for 12 weeks and all test products were shown to support long-term cell growth. PMID:2324238

Dahling, D R; Wright, B A

1990-03-01

61

A Biochemical Assay for Acetylcholinesterase Activity in PC12 Cells  

NSDL National Science Digital Library

This lab describes two biochemical assays: One for measuring acetylcholinesterase activity and one for measuring protein concentration. Students learn how to manipulate small-volume samples, use a standard spectrophotometer or a microplate reader spectrophotometer, construct a standard curve, and normalize data. The lab is intended to be used in conjunction with a cell culture lab in which PC12 cells are exposed to various agents that influence their phenotypic state.

Paul J. Schwartz (University of Medicine and Dentistry of New Jersey;Department of Neurological Surgery REV); Jay A. Blundon (Rhodes College;Department of Biology REV); Elizabeth M. Adler (American Association for the Advancement of Science;Science's STKE REV)

2007-07-10

62

Perfusion Based Cell Culture Chips  

NASA Astrophysics Data System (ADS)

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.

Heiskanen, A.; Emnéus, J.; Dufva, M.

63

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

PubMed Central

Objective(s): This study aimed to find out whether the chick cardiomyocyte micromass (MM) system could be employed to predict the teratogenecity of common environmental factors. Different multivitamins and over the counter drugs were used in this study. Materials and Methods: White Leghorn 5-day-old embryo hearts were dissected and trypsinized to produce a cardiomyocyte cell suspension in Dulbecco's Modified Eagle's Medium. The cultures were incubated at 370C in 5% CO2 in air, and observations were made at 24, 48 and 144 hr, for the detection of cell beating. Cellular viability was assessed using the resazurin assay and cell protein content was assessed by the kenacid blue assay. It was observed that while not affecting total cell number folic acid, vitamin C, sodium fluoride and ginseng did not significantly reduced cell activity and beating. However cadmium chloride significantly reduced the beating, cell viability and cell protein content in micromass cultures. Results: The results demonstrate the potential of the chick cardiomyocyte MM culture assay to identify teratogens/embryotoxins that alter morphology and function, which may result in either teratogenic outcome or cytotoxicity. Conclusion: This could form part of a screen for developmental toxicity related to cardiac function. PMID:24171079

Memon, Samreen; Pratten, Margaret

2013-01-01

64

Culture of organized cell communities  

Microsoft Academic Search

Cells cultured in vitro will tend to retain their differentiated phenotype under conditions that resemble their natural in vivo environment, for example, when cultured on polymer scaffolds in tissue culture bioreactors. In this chapter, we define organized cell communities as three-dimensional in vitro grown cell–polymer constructs that display important structural and functional features of the natural tissue. We review representative

Lisa E. Freed; Gordana Vunjak-Novakovic

1998-01-01

65

Evaluation of The Alamarblue Assay for Adherent Cell Irradiation Experiments  

PubMed Central

The AlamarBlue assay is based on fluorometric detection of metabolic mitochondrial activity of cells. In this study, we determined the methodology for application of the assay to radiation response experiments in 96-well plates. AlamarBlue was added and its reduction measured 7 hours later. Selection of the initial number of plated cells was important so that the number of proliferating cells remains lower than the critical number that produced full AlamarBlue reduction (plateau phase) at the time points of measurements. Culture medium was replaced twice a week to avoid suppression of viability due to nutrient competition and metabolic waste accumulation. There was no need to replace culture medium before adding AlamarBlue. Cell proliferation continued after irradiation and the suppression effect on cell viability was most evident on day 8. At this time point, by comparing measurements from irradiated vs. non-irradiated cells, for various dose levels, a viability dose response curve was plotted. Immediately after the 8th day (nadir), cells started to re-grow at a rate inversely related to the radiation dose. By comparing measurements at the time point of nadir vs. a convenient subsequent time point, re-growth dose response abilities were plotted, simulating clonogenic assays. PMID:24910583

Zachari, Maria A.; Chondrou, Panagiota S.; Pouliliou, Stamatia E.; Mitrakas, Achilleas G.; Abatzoglou, Ioannis; Zois, Christos E.; Koukourakis, Michael I.

2014-01-01

66

Fluorometric assay for red blood cell antibodies  

SciTech Connect

A fluorometric assay is described for the detection of red blood cell antibodies. The assay reveals as little as 600 molecules of bound, fluoroesceinated rabbit anti-human IgG antibodies per erythrocyte. Eleven patients with possible autoimmune erythrocyte disorder and negative direct antiglobulin test were studied by the fluorometric assay. The outcome of the fluorometric assay was compared with that of the human allogeneic rosette test. Results obtained by the two methods were in complete agreement. Five of the patients were shown to possess unexpectedly high levels of erythrocyte-bound IgG in spite of a negative, direct antiglobulin test. These findings and the validity of the fluorometric assay are discussed.

Schreiber, A.B.; Lambermont, M.; Strosberg, A.D.; Wybran, J.

1981-03-01

67

Cell death assays for drug discovery  

Microsoft Academic Search

Cell death has an important role in many human diseases, and strategies aimed at modulating the associated pathways have been successfully applied to treat various disorders. Indeed, several clinically promising cytotoxic and cytoprotective agents with potential applications in cancer, ischaemic and neurodegenerative diseases have recently been identified by high-throughput screening (HTS), based on appropriate cell death assays. Given that different

Oliver Kepp; Lorenzo Galluzzi; Marta Lipinski; Junying Yuan; Guido Kroemer

2011-01-01

68

Fluorescence Assay 2. http://www.tgrbio.com/cancer-cell-lines-primary-cell-  

E-print Network

Fluorescence Assay References 1. 2. http://www.tgrbio.com/cancer-cell-lines-primary-cell- cultures). It is the principle target receptor for the relief of nausea and vomiting caused by chemo- and radio-therapies in cancer patients. This makes the study of both agonist and antagonist ligands important as the knowledge

Collins, Gary S.

69

A plate reader-compatible microchannel array for cell biology assays{ Hongmei Yu,a  

E-print Network

A plate reader-compatible microchannel array for cell biology assays{ Hongmei Yu,a Caroline M in the life sciences. Here, we present a microchannel cell culture platform having both operational cells (both a cell line and primary cells) in the microchannel arrays. The utilization of ubiquitous

Beebe, David J.

70

In Vivo Assay for Tumor Cell Invasion  

PubMed Central

Summary We describe an in vivo invasion assay that enables the collection of invasive cells from the primary tumor. In addition to determination of the endogenous, unstimulated invasive properties of cells in vivo, the assay can take advantage of the chemotactic properties of cancer cells. Microneedles are filled with a mixture of extracellular matrix components such as Matrigel with or without a chemoattractant such as EGF, and then introduced into the primary tumor of a rat or mouse that is generated either by orthotopic injection of carcinoma cell lines or by a transgene such as polyoma Middle T. Over the course of 4 h the invasive cell population enters the needles while the animal is kept under anesthesia. At the end of the collection time, the invasive cells are extruded from the microneedles and can be analyzed in terms of the number and type of cells that invade in response to defined stimuli. By including pharmacological inhibitors in the needle, signaling pathways contributing to in vivo invasion can also be identified. This assay leads to a better understanding of the cell types and signaling involved in the tumor microenvironment, and has the potential to be applied to a variety of in vivo models. PMID:19763970

Hernandez, Lorena; Smirnova, Tatiana; Wyckoff, Jeffrey; Condeelis, John; Segall, Jeffrey E.

2010-01-01

71

Polyester ?-assay chip for stem cell studies  

PubMed Central

The application of microfluidic technologies to stem cell research is of great interest to biologists and bioengineers. This is chiefly due to the intricate ability to control the cellular environment, the reduction of reagent volume, experimentation time and cost, and the high-throughput screening capabilities of microscale devices. Despite this importance, a simple-to-use microfluidic platform for studying the effects of growth factors on stem cell differentiation has not yet emerged. With this consideration, we have designed and characterized a microfluidic device that is easy to fabricate and operate, yet contains several functional elements. Our device is a simple polyester-based microfluidic chip capable of simultaneously screening multiple independent stem cell culture conditions. Generated by laser ablation and stacking of multiple layers of polyester film, this device integrates a 10?×?10 microwell array for cell culture with a continuous perfusion system and a non-linear concentration gradient generator. We performed numerical calculations to predict the gradient formation and calculate the shear stress acting on the cells inside the device. The device operation was validated by culturing murine embryonic stem cells inside the microwells for 5 days. Furthermore, we showed the ability to maintain the pluripotency of stem cell aggregates in response to concentrations of leukemia inhibitory factor ranging from 0 to ?1000 U/ml. Given its simplicity, fast manufacturing method, scalability, and the cell-compatible nature of the device, it may be a useful platform for long-term stem cell culture and studies. PMID:24278097

Piraino, Francesco; Selimovi?, Šeila; Adamo, Marco; Pero, Alessandro; Manoucheri, Sam; Bok Kim, Sang; Demarchi, Danilo; Khademhosseini, Ali

2012-01-01

72

Cell culture purity issues and DFAT cells  

SciTech Connect

Highlights: •DFAT cells are progeny cells derived from dedifferentiated mature adipocytes. •Common problems in this research is potential cell contamination of initial cultures. •The initial cell culture purity is crucial in DFAT cell research field. -- Abstract: Dedifferentiation of mature adipocytes, in vitro, has been pursued/documented for over forty years. The subsequent progeny cells are named dedifferentiated adipocyte-derived progeny cells (DFAT cells). DFAT cells are proliferative and likely to possess mutilineage potential. As a consequence, DFAT cells and their progeny/daughter cells may be useful as a potential tool for various aspects of tissue engineering and as potential vectors for the alleviation of several disease states. Publications in this area have been increasing annually, but the purity of the initial culture of mature adipocytes has seldom been documented. Consequently, it is not always clear whether DFAT cells are derived from dedifferentiated mature (lipid filled) adipocytes or from contaminating cells that reside in an impure culture.

Wei, Shengjuan [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi Province 712100 (China) [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi Province 712100 (China); Department of Animal Sciences, Washington State University, Pullman, WA 99164 (United States); Bergen, Werner G. [Program in Cellular and Molecular Biosciences/Department of Animal Sciences, Auburn University, Auburn, AL 36849 (United States)] [Program in Cellular and Molecular Biosciences/Department of Animal Sciences, Auburn University, Auburn, AL 36849 (United States); Hausman, Gary J. [Animal Science Department, University of Georgia, Athens, GA 30602-2771 (United States)] [Animal Science Department, University of Georgia, Athens, GA 30602-2771 (United States); Zan, Linsen, E-mail: zanls@yahoo.com.cn [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi Province 712100 (China)] [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi Province 712100 (China); Dodson, Michael V., E-mail: dodson@wsu.edu [Department of Animal Sciences, Washington State University, Pullman, WA 99164 (United States)

2013-04-12

73

Oxygen Control For Bioreactors And In-vitro Cell Assays  

NASA Astrophysics Data System (ADS)

Dissolved oxygen (DO) is an important parameter in biomedical and cell-culture applications. Several studies have found cell survival and function to be intimately linked to oxygen concentration. Laminar flow, as observed in microfluidic devices, provides an ideal environment to manipulate and control concentration gradients. In this paper we demonstrate the first characterization of integrated fluorescence-based oxygen sensors for DO measurement within a cell-culture bioreactor device. Solid-state PtOEPK/PS sensor patterns were integrated into the PDMS-based bioreactor and calibrated for detection of DO concentration with a superimposed layer of collagen and Ishikawa human endometrial cancer cells. The sensor signal of the layer subjacent to the cells was found to follow a Stern-Volmer model and the intensity ratio was measured to I0/I100 = 3.9 after 3 days in culture. The device provides a novel tool for the control and spatially-resolved measurement of oxygen levels in cellular assays and cell-culture applications.

Nock, V.; Blaikie, R. J.; David, T.

2009-07-01

74

Adaptive Active Classification of Cell Assay Images  

E-print Network

Adaptive Active Classification of Cell Assay Images Nicolas Cebron and Michael R. Berthold ALTANA propose a new adaptive active classification scheme which estab- lishes ties between the two opposing concepts of unsupervised clustering of the underlying data and the supervised task of classification. Based

Reiterer, Harald

75

DETECTION AND TITRATION OF BLUETONGUE VIRUS IN CULICOIDES INSECT CELL CULTURE BY AN ANTIGEN-CAPTURE ENZYME-LINKED IMMUNOSORBENT ASSAY  

Technology Transfer Automated Retrieval System (TEKTRAN)

Bluetongue virus (BTV) infects sheep, cattle and other ruminants and is transmitted by Culicoides spp. of biting midges. Virus is typically isolated and characterized by infection of susceptible vertebrate cells that undergo detectable and measurable cytopathic effects. Cell lines derived from C. ...

76

Isolation and Culture of Epithelial Stem Cells  

PubMed Central

In the skin, epithelial stem cells in the hair follicle contribute not only to the generation of a new hair follicle with each hair cycle, but also to the repair of the epidermis during wound healing. When these stem cells are isolated and expanded in culture, they can give rise to hair follicles, sebaceous glands, and epidermis when combined with dermis and grafted back onto Nude mice. In this chapter, we provide a method for isolating hair follicle epithelial stem cells from the skin of adult mice using immunofluorescent labeling to allow for the specific purification of epithelial stem cells by fluorescence-activated cell sorting (FACS). Notably, this method relies exclusively on cell surface markers, making it suitable for use with any strain of mouse and at various stages of the hair cycle. We also provide a detailed protocol for culturing epithelial stem cells isolated by FACS, allowing for analysis using a wide variety of culture assays. Additionally, we provide notes on using cultured cells for specific applications, such as viral manipulation and grafting. These techniques should be useful for directly evaluating stem cell function in normal mice and in mice with skin defects. PMID:19089359

Nowak, Jonathan A.; Fuchs, Elaine

2009-01-01

77

Culture of Human Leukaemia Cells  

Microsoft Academic Search

THIS communication describes the culture of four additional cell lines derived from the buffy coats of patients with leukaemia. Iwakata and Grace1 provided key information for culturing leukaemia cells and reported the establishment of a cell line, R.P.M.I. No. 6410. Fifteen cell lines were subsequently derived from the buffy coats of four patients with acute and chronic myelocytic leukaemia and

G. E. Moore; E. Ito; P. Citron

1966-01-01

78

High-ThroughputHigh-Throughput Cell Death Cell Death AssaysCell Death Assays.  

PubMed

High-throughput screensHigh throughput screens (HTSHTS ) are powerful tools that permit the rapid evaluation of thousands of samplessamples in a cost-effective manner and minimize samplesample and reagent consumption. Such assaysassays have recently begun to be utilized to evaluate cell deathcell death modalities and also the cytoprotectivecytoprotective efficacycytoprotective efficacy of compounds against a wide variety of stresses. Here we describe the design, preparation, and undertaking of HTS-appropriate assays that utilize simple and cost-effective fluorophorefluorophore - and luminescenceluminescence -based functional readouts of cell viability. These assays permit the examination of 96-384 compounds in a single multiwell platemultiwell plate with highly robust statistical significancestatistical significance at a fraction of the financial and work cost of traditional approaches. PMID:25431064

Pamenter, Matthew E; Haddad, Gabriel G

2015-01-01

79

High density cell culture system  

NASA Technical Reports Server (NTRS)

An annular culture vessel for growing mammalian cells is constructed in a one piece integral and annular configuration with an open end which is closed by an endcap. The culture vessel is rotatable about a horizontal axis by use of conventional roller systems commonly used in culture laboratories. The end wall of the endcap has tapered access ports to frictionally and sealingly receive the ends of hypodermic syringes. The syringes permit the introduction of fresh nutrient and withdrawal of spent nutrients. The walls are made of conventional polymeric cell culture material and are subjected to neutron bombardment to form minute gas permeable perforations in the walls.

Spaulding, Glenn F. (inventor)

1994-01-01

80

21 CFR 864.7100 - Red blood cell enzyme assay.  

...2014-04-01 2014-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure...

2014-04-01

81

21 CFR 864.7100 - Red blood cell enzyme assay.  

Code of Federal Regulations, 2010 CFR

...2010-04-01 2010-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure...

2010-04-01

82

21 CFR 864.7100 - Red blood cell enzyme assay.  

Code of Federal Regulations, 2013 CFR

...2013-04-01 2013-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure...

2013-04-01

83

21 CFR 864.7100 - Red blood cell enzyme assay.  

Code of Federal Regulations, 2011 CFR

...2011-04-01 2011-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure...

2011-04-01

84

21 CFR 864.7100 - Red blood cell enzyme assay.  

Code of Federal Regulations, 2012 CFR

...2012-04-01 2012-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure...

2012-04-01

85

Ovine Carotid Artery-Derived Cells as an Optimized Supportive Cell Layer in 2-D Capillary Network Assays  

PubMed Central

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 tissue cells. The use of ovine carotid artery-derived cells simplifies the endothelial co-culture assay with respect to testing large amounts of pro- and anti-angiogenic factors. PMID:24621607

Dreier, Agnieszka; Unger, Ronald E.; Flanagan, Thomas C.; Kirkpatrick, C. James; Zenke, Martin; Klee, Doris; Jockenhoevel, Stefan

2014-01-01

86

Novel application of a fish gill cell line assay to assess ichthyotoxicity of harmful marine microalgae  

Microsoft Academic Search

Fish-killing microalgae can cause substantial mortalities of cultured finfish, but their killing mechanisms are not completely understood. Since use of cell lines offers significant advantages compared to working with whole organisms, a simple in vitro assay for microalgal ichthyotoxicity is described using the rainbow trout cell line RTgill-W1. We describe the application of a microplate based assay for testing the

Juan José Dorantes-Aranda; T. David Waite; Aurélie Godrant; Andrew L. Rose; César D. Tovar; Gregory M. Woods; Gustaaf M. Hallegraeff

2011-01-01

87

Maintaining dendritic cell viability in culture.  

PubMed

When mouse dendritic cells (DCs) are isolated from tissues, purified and placed in a nutritive culture they die more rapidly than would be expected from their normal turnover in vivo. This can distort culture assays of DC function. We therefore tested several approaches to prolonging DC survival in culture. Of several cytokines tested granulocyte-macrophage colony stimulating factor was most effective at preserving the viability of conventional DCs (cDCs) but was ineffective for plasmacytoid DCs (pDCs). Surprisingly, Fms-like tyrosine kinase 3 ligand, crucial for DC development, produced only a marginal improvement in DC survival in culture, and interleukin-3, reported to prevent apoptosis of human pDCs, produced only a minor improvement in survival of mouse DCs. Genetic manipulation of cell death pathways was also tested, to avoid activation effects exerted by cytokine signalling. The isolation of DCs from mice overexpressing Bcl-2 was especially effective in maintaining pDC viability but gave a lesser improvement in cDC viability. DCs isolated from Bim(-/-)Noxa(-/-) mice also showed improved culture survival, but in this case with pDCs showing the least improvement. PMID:25081090

Vremec, David; Hansen, Jacinta; Strasser, Andreas; Acha-Orbea, Hans; Zhan, Yifan; O'Keeffe, Meredith; Shortman, Ken

2015-02-01

88

CYTOTOXICITY OF CHEMICAL CARCINOGENS TOWARDS HUMAN BRONCHIAL EPITHELIAL CELLS EVALUATED IN A CLONAL ASSAY  

EPA Science Inventory

Survival of human bronchial epithelial cells after administration of four chemical carcinogens was measured in a clonal assay. Human bronchial epithelial cells were obtained from outgrowths of explanted tissue pieces. Serum-free medium was used for both explant culture and clonal...

89

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

PubMed

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

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

2014-07-01

90

Shape Memory Polymers for Active Cell Culture  

PubMed Central

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape. Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture. Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions. PMID:21750496

Davis, Kevin A.; Luo, Xiaofan; Mather, Patrick T.; Henderson, James H.

2011-01-01

91

Shape memory polymers for active cell culture.  

PubMed

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat(1-5). In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, T(trans;) [either the melting temperature (T(m;)) or the glass transition temperature (T(g;))]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its T(trans;) while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through T(trans;) under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape. Cells are capable of surveying the mechanical properties of their surrounding environment(6). The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces(7-14). These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture. Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions. PMID:21750496

Davis, Kevin A; Luo, Xiaofan; Mather, Patrick T; Henderson, James H

2011-01-01

92

Aseptic technique for cell culture.  

PubMed

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

Coté, R J

2001-05-01

93

Development of a Drosophila Cell-Based Error Correction Assay  

PubMed Central

Accurate transmission of the genome through cell division requires microtubules from opposing spindle poles to interact with protein super-structures called kinetochores that assemble on each sister chromatid. Most kinetochores establish erroneous attachments that are destabilized through a process called error correction. Failure to correct improper kinetochore-microtubule (kt-MT) interactions before anaphase onset results in chromosomal instability (CIN), which has been implicated in tumorigenesis and tumor adaptation. Thus, it is important to characterize the molecular basis of error correction to better comprehend how CIN occurs and how it can be modulated. An error correction assay has been previously developed in cultured mammalian cells in which incorrect kt-MT attachments are created through the induction of monopolar spindle assembly via chemical inhibition of kinesin-5. Error correction is then monitored following inhibitor wash out. Implementing the error correction assay in Drosophila melanogaster S2 cells would be valuable because kt-MT attachments are easily visualized and the cells are highly amenable to RNAi and high-throughput screening. However, Drosophila kinesin-5 (Klp61F) is unaffected by available small molecule inhibitors. To overcome this limitation, we have rendered S2 cells susceptible to kinesin-5 inhibitors by functionally replacing Klp61F with human kinesin-5 (Eg5). Eg5 expression rescued the assembly of monopolar spindles typically caused by Klp61F depletion. Eg5-mediated bipoles collapsed into monopoles due, in part, to kinesin-14 (Ncd) activity when treated with the kinesin-5 inhibitor S-trityl-L-cysteine (STLC). Furthermore, bipolar spindles reassembled and error correction was observed after STLC wash out. Importantly, error correction in Eg5-expressing S2 cells was dependent on the well-established error correction kinase Aurora B. This system provides a powerful new cell-based platform for studying error correction and CIN. PMID:23888285

Salemi, Jeffrey D.; McGilvray, Philip T.; Maresca, Thomas J.

2013-01-01

94

Cultured Human Renal Cortical Cells  

NASA Technical Reports Server (NTRS)

During the STS-90 shuttle flight in April 1998, cultured renal cortical cells revealed new information about genes. Timothy Hammond, an investigator in NASA's microgravity biotechnology program was interested in culturing kidney tissue to study the expression of proteins useful in the treatment of kidney diseases. Protein expression is linked to the level of differentiation of the kidney cells, and Hammond had difficulty maintaining differentiated cells in vitro. Intrigued by the improvement in cell differentiation that he observed in rat renal cells cultured in NASA's rotating wall vessel (a bioreactor that simulates some aspects of microgravity) and during an experiment performed on the Russian Space Station Mir, Hammond decided to sleuth out which genes were responsible for controlling differentiation of kidney cells. To do this, he compared the gene activity of human renal cells in a variety of gravitational environments, including the microgravity of the space shuttle and the high-gravity environment of a centrifuge. Hammond found that 1,632 genes out of 10,000 analyzed changed their activity level in microgravity, more than in any of the other environments. These results have important implications for kidney research as well as for understanding the basic mechanism for controlling cell differentiation.

1998-01-01

95

Hematopoietic Stem Cells: Inferences-from In Vivo Assays  

E-print Network

Hematopoietic Stem Cells: Inferences-from In Vivo Assays CONNIEEAVES,CINDYMILLER,JOHANNE CASHMAN Columbia, Canada Key Words.Hematopoietic stem cells Transplantation Cord blood. Expansion Growthfactors murine hematopoietic stem cells to be quantitated. Measurements of murine CRU have shown

Zandstra, Peter W.

96

Principles of cancer cell culture.  

PubMed

The basics of cell culture are now relatively common, though it was not always so. The pioneers of cell culture would envy our simple access to manufactured plastics, media and equipment for such studies. The prerequisites for cell culture are a well lit and suitably ventilated laboratory with a laminar flow hood (Class II), CO(2) incubator, benchtop centrifuge, microscope, plasticware (flasks and plates) and a supply of media with or without serum supplements. Not only can all of this be ordered easily over the internet, but large numbers of well-characterised cell lines are available from libraries maintained to a very high standard allowing the researcher to commence experiments rapidly and economically. Attention to safety and disposal is important, and maintenance of equipment remains essential. This chapter should enable researchers with little prior knowledge to set up a suitable laboratory to do basic cell culture, but there is still no substitute for experience within an existing well-run laboratory. PMID:21516394

Cree, Ian A

2011-01-01

97

The XTT Cell Proliferation Assay Applied to Cell Layers Embedded in Three-Dimensional Matrix  

PubMed Central

Abstract Cell proliferation, a main target in cancer therapy, is influenced by the surrounding three-dimensional (3D) extracellular matrix (ECM). In vitro drug screening is, thus, optimally performed under conditions in which cells are grown (embedded or trapped) in dense 3D matrices, as these most closely mimic the adhesive and mechanical properties of natural ECM. Measuring cell proliferation under these conditions is, however, technically more challenging compared with two-dimensional (2D) culture and other “3D culture conditions,” such as growth on top of a matrix (pseudo-3D) or in spongy scaffolds with large pore sizes. Consequently, such measurements are only slowly applied on a wider scale. To advance this, we report on the equal quality (dynamic range, background, linearity) of measuring the proliferation of cell layers embedded in dense 3D matrices (collagen, Matrigel) compared with cells in 2D culture using the easy (one-step) and in 2D well-validated, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)-assay. The comparison stresses the differences in proliferation kinetics and drug sensitivity of matrix-embedded cells versus 2D culture. Using the specific cell-layer-embedded 3D matrix setup, quantitative measurements of cell proliferation and cell invasion are shown to be possible in similar assay conditions, and cytostatic, cytotoxic, and anti-invasive drug effects can thus be reliably determined and compared in physiologically relevant settings. This approach in the 3D matrix holds promise for improving early-stage, high-throughput drug screening, targeting either highly invasive or highly proliferative subpopulations of cancers or both. PMID:22574651

Huyck, Lynn; Ampe, Christophe

2012-01-01

98

Cell culture compositions  

DOEpatents

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.

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

2014-03-18

99

A novel coagulation assay incorporating adherent endothelial cells in thromboelastometry.  

PubMed

Following vascular injury or activation, endothelial cells (ECs) participate in the modulation of haemostasis and fibrinolysis. Viscoelastic tests (VETs) are a potent bedside monitoring tool that reports haemostatic parameters in real time. However, VETs neglect the influence of the surrounding endothelium. Our aim was therefore to establish an assay that incorporates ECs in a whole blood VET and to assess the impact of ECs on coagulation parameters. Outgrowth endothelial cells (OECs) and human umbilical vein endothelial cells (HUVECs) were seeded onto microbeads to create transferable EC-microcarriers. Microbeads were then added to citrated whole blood in the measurement cup of a thromboelastometry device (ROTEM). After the addition of CaCl2 (star-TEM®) to the blood sample (NATEM assay), standard ROTEM parameters were analysed. Scanning electron microscopy (SEM) was carried out to visualise the interactions of the beads, whole blood components and the ROTEM pin after clotting. SEM showed that the added microbeads were effectively incorporated into the final blood clot. In the presence of activated ECs, the clotting time (CT) of the blood was shortened fourfold compared to that in uncoated control beads. A significant reduction in CT was also observed in the presence of unstimulated ECs. Interestingly, CT was also reduced by the addition of purified EC culture supernatant. CT shortening was prevented by incubating the supernatant with an inhibiting antibody against tissue factor (TF). Our findings demonstrate that ECs can be incorporated into a ROTEM assay via coated microbeads, and whole blood clotting initiation is accelerated by non-activated and activated ECs. PMID:23494019

Zipperle, J; Schlimp, C J; Holnthoner, W; Husa, A-M; Nürnberger, S; Redl, H; Schöchl, H

2013-05-01

100

Hawthorn (Crataegus monogyna Jacq.) extract exhibits atropine-sensitive activity in a cultured cardiomyocyte assay.  

PubMed

Hawthorn (Crataegus spp.) plant extract is used as a herbal alternative medicine for the prevention and treatment of various cardiovascular diseases. Recently, it was shown that hawthorn extract preparations caused negative chronotropic effects in a cultured neonatal murine cardiomyocyte assay, independent of beta-adrenergic receptor blockade. The aim of this study was to further characterize the effect of hawthorn extract to decrease the contraction rate of cultured cardiomyocytes. To test the hypothesis that hawthorn is acting via muscarinic receptors, the effect of hawthorn extract on atrial versus ventricular cardiomyocytes in culture was evaluated. As would be expected for activation of muscarinic receptors, hawthorn extract had a greater effect in atrial cells. Atrial and/or ventricular cardiomyocytes were then treated with hawthorn extract in the presence of atropine or himbacine. Changes in the contraction rate of cultured cardiomyocytes revealed that both muscarinic antagonists significantly attenuated the negative chronotropic activity of hawthorn extract. Using quinuclidinyl benzilate, L-[benzylic-4,4'-(3)H] ([(3)H]-QNB) as a radioligand antagonist, the effect of a partially purified hawthorn extract fraction to inhibit muscarinic receptor binding was quantified. Hawthorn extract fraction 3 dose-dependently inhibited [(3)H]-QNB binding to mouse heart membranes. Taken together, these findings suggest that decreased contraction frequency by hawthorn extracts in neonatal murine cardiomyocytes may be mediated via muscarinic receptor activation. PMID:18696181

Salehi, Satin; Long, Shannon R; Proteau, Philip J; Filtz, Theresa M

2009-01-01

101

Versatile, Fully Automated, Microfluidic Cell Culture System  

E-print Network

Versatile, Fully Automated, Microfluidic Cell Culture System Rafael Go´mez-Sjo1berg, Anne A. Leyrat and quantita- tive cell culture technology, driven both by the intense activity in stem cell biology and by the emergence of systems biology. We built a fully automated cell culture screening system based

Chen, Christopher S.

102

Ocular irritation reversibility assessment for personal care products using a porcine corneal culture assay.  

PubMed

Personal care product manufacturers have used a broad spectrum of alternative ocular irritation assays during the past two decades because these tests do not require the use of live animals, they provide reliable predictive data, and they are relatively inexpensive to conduct. To complement these assays, the ex vivo Porcine Corneal Opacity Reversibility Assay (PorCORA) was recently developed using a corneal culture model to predict reversibility of ocular irritants. Three commercially available consumer products (a shampoo, a hair color glaze, and a hair colorant system containing 12% hydrogen peroxide) were each tested in two PorCORA study replicates in order to assess potential ocular damage reversibility for surfactant-, propylene carbonate-, and peroxide-based formulations, respectively. Under the exaggerated, in vitro study conditions, the surfactant-based shampoo may cause irreversible porcine corneal damage (histological changes in the epithelial squamous cell and/or basal cell layers), whereas the hair color glaze and 12% hydrogen peroxide product caused fully reversible ocular irritation (microscopic changes only in the superficial squamous cell layer). The hair color glaze and peroxide product results correlate with established in vivo data for similar compounds, but the shampoo results contradicted previous BCOP results (expected to be only a mild irritant). Therefore, although the PorCORA protocol shows promise in predicting the extent and reversibility of potential ocular damage caused by accidental consumer eye exposure to personal care products, the contradictory results for the surfactant-based shampoo indicate that more extensive validation testing of the PorCORA is necessary to definitively establish the protocol's reliability as a Draize test replacement. PMID:21172418

Donahue, Douglas A; Avalos, Javier; Kaufman, Lewis E; Simion, F Anthony; Cerven, Daniel R

2011-04-01

103

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

Microsoft Academic Search

Twenty-seven chemicals were tested for their mutagenic potential in the L5178Y tk{sup +}\\/tk⁻ 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 μg\\/ml. The chemicals were tested at least twice. Statistically significant responses were obtained with acid orange

Douglas B. McGregor; Alison G. Brown; Susan Howgate; Douglas McBride; Colin Riach; William J. Caspary; J. H. Carver

1991-01-01

104

Resistance to lipid peroxidation by cultured neoplastic cells  

SciTech Connect

The membranes of murine neuroblastoma cells (C1300) and human leukemia cells (HL-60) exhibit markedly increased resistance to peroxidation and undifferentiated Friend erythroleukemia cells were highly resistant to peroxidation. These findings suggest that high resistance to peroxidation and changes in the level of resistance occur commonly in cultured cells. Both cytosolic and membrane-associated factors that can prevent the onset of lipid peroxidation are present in differentiating neuroblastoma cells. A highly sensitive, single-phase assay for antioxidant activity failed to detect the presence of an antioxidant that could be associated with increased resistance to peroxidation in neuroblastoma cells. Likewise, lipid analyses of neuroblastoma cells revealed no parameter that could be related to this increase; however, this resistance phenomenon is abolished by adding arachidonic acid to the culture medium at levels that do not affect cell growth or viability. Protective factors exist in the cytosolic fraction of rat liver homogenate, which are able to neutralize the toxic products of lipid peroxidation rather than prevent the initiation of peroxidation. These protective factors were detected, and could possibly be isolated, by a cytotoxicity assay employing Chinese hamster ovary cells. In the course of this work, we discovered an antioxidant artifact that is widely distributed in commercial tissue culture media. A simple procedure has been developed to detect this antioxidant in lots of culture media.

Arneson, R.M.; Wander, J.D.; Cabot, M.C.; Tan, E.L.; Schenley, R.L.; Hsie, A.W.

1982-01-01

105

Simultaneous isolation of enriched myoblasts and fibroblasts for migration analysis within a novel co-culture assay.  

PubMed

Skeletal muscle injury elicits the activation of satellite cells and their migration to the wound area for subsequent terminal differentiation and tissue integration. However, interstitial fibroblasts recruited to the site of injury promote deposition of fibrotic tissue, which hampers myoblast-mediated muscle regeneration. Currently, analysis of myoblast migration in vitro can be accomplished using chemotactic, cell-exclusion, or wound healing assays. Yet, to investigate cell motility following skeletal muscle damage more accurately, migration assays need to better simulate the repair process. Here we present a protocol for the simultaneous isolation of myoblasts and fibroblasts from the same muscle tissue, ensuring the consistent generation of enriched, purified, and matched cell populations at a low passage number. We then describe a wound assay that uses a novel approach to the co-culture of myoblasts and fibroblasts to mimic the injured environment more closely than other established methods. Using this assay, we demonstrate that fibroblasts are able to increase myoblast migration significantly, validating our new in vitro method. As the observed effect on migration is most likely mediated by secreted factors, our assay could easily be extended to include antibody-based protein analysis of secreted factors in animal or human systems. PMID:25605577

Goetsch, Kyle Peter; Snyman, Celia; Myburgh, Kathryn Helen; Niesler, Carola Ulrike

2014-01-01

106

9 CFR 101.6 - Cell cultures.  

Code of Federal Regulations, 2010 CFR

...2010-01-01 2010-01-01 false Cell cultures. 101.6 Section 101.6...AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to...

2010-01-01

107

9 CFR 101.6 - Cell cultures.  

Code of Federal Regulations, 2013 CFR

...2013-01-01 2013-01-01 false Cell cultures. 101.6 Section 101.6...AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to...

2013-01-01

108

9 CFR 101.6 - Cell cultures.  

...2014-01-01 2014-01-01 false Cell cultures. 101.6 Section 101.6...AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to...

2014-01-01

109

9 CFR 101.6 - Cell cultures.  

Code of Federal Regulations, 2012 CFR

...2012-01-01 2012-01-01 false Cell cultures. 101.6 Section 101.6...AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to...

2012-01-01

110

9 CFR 101.6 - Cell cultures.  

Code of Federal Regulations, 2011 CFR

...2011-01-01 2011-01-01 false Cell cultures. 101.6 Section 101.6...AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to...

2011-01-01

111

Mammosphere culture of cancer stem cells in a microfluidic device  

NASA Astrophysics Data System (ADS)

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.

Saadin, Katayoon; White, Ian M.

2012-03-01

112

Opsonophagocytic assay.  

PubMed

The opsonophagocytic killing (OPK) assay is used as a correlate for protection to measure the functional capacities of vaccine-candidate-raised antibodies. This in vitro assay aids selecting promising vaccines by demonstrating whether the vaccine-induced antibodies drive efficient complement deposition and subsequent opsonophagocytic killing. Here, we describe two protocols for an OPK assay using either human-derived PMNs or cultured HL-60 cells. PMID:24218277

Dwyer, Markryan; Gadjeva, Mihaela

2014-01-01

113

Multiwell stiffness assay for the study of cell responsiveness to cytotoxic drugs  

PubMed Central

It is now well understood that the cell microenvironment, including the surrounding matrix, profoundly affects cell fate. This is especially true for solid tumors where, for example, matrix stiffness is believed to be an important factor in tumorogenesis. Our hypothesis is that since matrix stiffness affects cell fate, it may also be important in drug resistance. To test this hypothesis, we designed and built a multiwell polyacrylamide (PA) gel-based stiffness assay, in which the gels were coated with collagen in order to facilitate cell attachment and proliferation. This PA-based assay was used to examine the effect of stiffness on cultured cell responsiveness to cytotoxic drugs. In particular, we tested multiple cancer cell lines and their susceptibility to paclitaxel, a microtubule-targeting agent. By assessing cell proliferation, morphology, and the IC50 of the drug, we were able to establish that the stiffness affects responsiveness to cytotoxic drugs in a cell dependent manner. PMID:24018833

Zustiak, Silviya; Nossal, Ralph; Sackett, Dan L.

2013-01-01

114

Multizone Paper Platform for 3D Cell Cultures  

PubMed Central

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. PMID:21573103

Derda, Ratmir; Hong, Estrella; Mwangi, Martin; Mammoto, Akiko; Ingber, Donald E.; Whitesides, George M.

2011-01-01

115

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice  

PubMed Central

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage. PMID:24637893

Walker, Tara L.; Kempermann, Gerd

2014-01-01

116

Electrolytic valving isolation of cell co-culture microenvironment with controlled cell pairing ratios.  

PubMed

Cancer-stromal interaction is a critical process in tumorigenesis. Conventional dish-based co-culture assays simply mix two cell types in the same dish; thus, they are deficient in controlling cell locations and precisely tracking single cell behavior from heterogeneous cell populations. Microfluidic technology can provide a good spatial-temporal control of microenvironments, but the control has been typically realized by using external pumps, making long-term cultures cumbersome and bulky. In this work, we have presented a cell-cell interaction microfluidic platform that can accurately control the co-culture microenvironment by using a novel electrolytic cell isolation scheme without using any valves or pneumatic pumps. The proposed microfluidic platform can also precisely control the number of interacting cells and pairing ratios to emulate cancer niches. More than 80% of the chambers captured the desired number of cells. The duration of cell isolation can be adjusted by electrolytic bubble generation and removal. We have verified that the electrolytic process has a negligible effect on cell viability and proliferation in our platform. To the best of our knowledge, this work is the first attempt to incorporate electrolytic bubble generation as a cell isolation method in microfluidics. For proof of feasibility, we have performed cell-cell interaction assays between prostate cancer (PC3) cells and myoblast (C2C12) cells. The preliminary results demonstrated the potential of using electrolysis for micro-environmental control during cell culture. Also, the ratio controlled cell-cell interaction assays were successfully performed which showed that the cell pairing ratios of PC3 to C2C12 affected the proliferation rate of myoblast cells due to increased secretion of growth factors from prostate cancer cells. PMID:25118341

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

2014-12-21

117

Culture and differentiation of embryonic stem cells  

Microsoft Academic Search

Summary Techniques are described for the culture of murine embryonic stem cells in the absence of heterologous feeder cells and for the induction of differentiation programs. The regulatory factor differentiation inhibiting activity\\/ leukaemia inhibitory factor (DIA\\/LIF) is produced at high concentration by transient expression in Cos cells and is used to suppress stem cell differentiation by addition to the culture

Austin G. Smith

1991-01-01

118

Miniature Bioreactor System for Long-Term Cell Culture  

NASA Technical Reports Server (NTRS)

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.

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

2010-01-01

119

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates  

PubMed Central

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates. The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival. The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens. PMID:19066533

Frank, Lauren E.; Caldera-Siu, Angela D.; Pothos, Emmanuel N.

2008-01-01

120

Bruce Walcheck: Role of neutrophils in acute lung injury, bacterial lung infection, and sepsis (cell based assays and in vivo studies in mice).  

E-print Network

cultures of primary keratinocytes from normal and atopic dermatitis dogs to test the effects of drugs Larson: Role of mast cells in the brain in the regulation of pain (cell based assays and in vivo studies in mice and rats). Al Beitz: Cancer inflammation and effects on pain (cell based assays and in vivo

Blanchette, Robert A.

121

Molecular Predictors of 3D Morphogenesis by Breast Cancer Cell Lines in 3D Culture  

E-print Network

Molecular Predictors of 3D Morphogenesis by Breast Cancer Cell Lines in 3D Culture Ju Han1 , Hang to triple-negative pathobiology. PPARc has been validated through two supporting biological assays. Citation by Breast Cancer Cell Lines in 3D Culture. PLoS Comput Biol 6(2): e1000684. doi:10.1371/journal.pcbi.1000684

Kenny, Paraic

122

DETECTION OF ANEUPLOIDY BY A MONOCHROMOSOMAL HYBRID CELL ASSAY  

EPA Science Inventory

A short-term assay utilizing human/mouse monochromosomal hybrid cells to detect chemically-induced aneuploidy in mammalian cells is described. A single human chromosome transferred into mouse cells was used as a cytogenetic marker to quantitate abnormal chromosome segregation fol...

123

Epithelial cells as alternative human biomatrices for comet assay  

PubMed Central

The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases. PMID:25506353

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

2014-01-01

124

Comet assay, cloning assay, and light and electron microscopy on one preselected cell  

NASA Astrophysics Data System (ADS)

In order to perform long-term studies up to one week on a preselected single cell after micromanipulation (e.g. UVA and NIR microbeam exposure) in comparison with non-treated neighbor cells (control cells) we applied a variety of single cell diagnostic techniques and developed a special comet assay for single preselected cells. For that purpose adherent cells were grown in low concentrations and maintained in special sterile centimeter-sized glass cell chambers. After preselection, a single cell was marked by means of diamond-produced circles on the outer cell chamber window. During exposure to microbeams, NADH-attributed autofluorescence of the chosen cell was detected by fluorescence imaging and spectroscopy. In addition, cell morphology was video-monitored (formation of pseudopodia, membrane blebbing,...). Maintaining the microchamber in the incubator, the irradiated cell was examined 24 h later for cell division (clone formation) and modifications in autofluorescence and morphology (including daughter cells). In the case that no division occurred the vitality of the light-exposed cell and of the control cells were probed by intranuclear propidium iodide accumulation. After fixation, either electron microscopy or single cell gel electrophoresis (comet assay) was performed. To monitor comet formation indicating photoinduced DNA damage in the preselected single cell in comparison with the non-exposed neighbor cells the chamber was filled with low-melting gel and lysis solution and exposed to an electric field. In contrast to the conventional comet assay, where only randomly chosen cells of a suspension are investigated, the novel optimized electrophoresis technique should enhance the possibilities of DNA damage detection to a true single (preselected) cell level. The single cell techniques applied to UVA microexposed Chinese hamster ovary cells (364 nm, 1 mW, 3.5 W/cm2) revealed significant cell damage for J/cm2 fluences such as modifications of intracellular redox state, impaired cell division, formation of giant cells and cell shrinking, swelling of mitochondria and loss of cristae as well as DNA damage.

Koenig, Karsten; Oehring, H.; Halbhuber, Karl-Juergen; Fiedler, Ursula; Bauer, Eckhard; Greulich, Karl O.

1997-12-01

125

Comet assay, cloning assay, and light and electron microscopy on one preselected cell  

NASA Astrophysics Data System (ADS)

In order to perform long-term studies up to one week on a preselected single cell after micromanipulation (e.g. UVA and NIR microbeam exposure) in comparison with non-treated neighbor cells (control cells) we applied a variety of single cell diagnostic techniques and developed a special comet assay for single preselected cells. For that purpose adherent cells were grown in low concentrations and maintained in special sterile centimeter-sized glass cell chambers. After preselection, a single cell was marked by means of diamond-produced circles on the outer cell chamber window. During exposure to microbeams, NADH-attributed autofluorescence of the chosen cell was detected by fluorescence imaging and spectroscopy. In addition, cell morphology was video-monitored (formation of pseudopodia, membrane blebbing,...). Maintaining the microchamber in the incubator, the irradiated cell was examined 24 h later for cell division (clone formation) and modifications in autofluorescence and morphology (including daughter cells). In the case that no division occurred the vitality of the light-exposed cell and of the control cells were probed by intranuclear propidium iodide accumulation. After fixation, either electron microscopy or single cell gel electrophoresis (comet assay) was performed. To monitor comet formation indicating photoinduced DNA damage in the preselected single cell in comparison with the non-exposed neighbor cells the chamber was filled with low-melting gel and lysis solution and exposed to an electric field. In contrast to the conventional comet assay, where only randomly chosen cells of a suspension are investigated, the novel optimized electrophoresis technique should enhance the possibilities of DNA damage detection to a true single (preselected) cell level. The single cell techniques applied to UVA microexposed Chinese hamster ovary cells (364 nm, 1 mW, 3.5 W/cm2) revealed significant cell damage for J/cm2 fluences such as modifications of intracellular redox state, impaired cell division, formation of giant cells and cell shrinking, swelling of mitochondria and loss of cristae as well as DNA damage.

Koenig, Karsten; Oehring, Hartmut; Halbhuber, Karl-Juergen; Fiedler, Ursula; Bauer, Eckhard; Greulich, Karl-Otto

1998-01-01

126

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

SciTech Connect

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.

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

1983-10-01

127

Microfluidic devices for studying heterotypic cell-cell interactions and tissue specimen cultures under controlled microenvironments  

PubMed Central

Microfluidic devices allow for precise control of the cellular and noncellular microenvironment at physiologically relevant length- and time-scales. These devices have been shown to mimic the complex in vivo microenvironment better than conventional in vitro assays, and allow real-time monitoring of homotypic or heterotypic cellular interactions. Microfluidic culture platforms enable new assay designs for culturing multiple different cell populations and?or tissue specimens under controlled user-defined conditions. Applications include fundamental studies of cell population behaviors, high-throughput drug screening, and tissue engineering. In this review, we summarize recent developments in this field along with studies of heterotypic cell-cell interactions and tissue specimen culture in microfluidic devices from our own laboratory. PMID:21522496

Zervantonakis, Ioannis K.; Kothapalli, Chandrasekhar R.; Chung, Seok; Sudo, Ryo; Kamm, Roger D.

2011-01-01

128

Assaying endothelial-mural cell interactions  

Technology Transfer Automated Retrieval System (TEKTRAN)

Recent studies in genetically malleable embryonic model systems have enabled the identification of factors required for blood vessel formation. However, it is not possible in most in vivo systems to dissect carefully the exact cellular behaviours, as well as cell-cell and cell-matrix interactions t...

129

Cell Culture as an Alternative in Education.  

ERIC Educational Resources Information Center

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)

Nardone, Roland M.

1990-01-01

130

Cell culture techniques in honey bee research  

Technology Transfer Automated Retrieval System (TEKTRAN)

Cell culture techniques are indispensable in most if not all life science disciplines to date. Wherever cell culture models are lacking scientific development is hampered. Unfortunately this has been and still is the case in honey bee research because permanent honey bee cell lines have not yet been...

131

Cell Culture Models of Transmissible Spongiform Encephalopathies  

Microsoft Academic Search

In this review, we describe the generation and use of cell culture models of transmissible spongiform encephalopathies, also known as prion diseases. These models include chronically prion-infected cell lines, as well as cultures expressing variable amounts of wild-type, mutated, or chimeric prion proteins. These cell lines have been widely used to investigate the biology of both the normal and the

Florence Béranger; Alain Mangé; Jérôme Solassol; Sylvain Lehmann

2001-01-01

132

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

PubMed Central

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. PMID:24511147

Yamauchi, Motohiro; Otsuka, Kensuke; Kondo, Hisayoshi; Hamada, Nobuyuki; Tomita, Masanori; Takahashi, Masayuki; Nakasono, Satoshi; Iwasaki, Toshiyasu; Yoshida, Kazuo

2014-01-01

133

Cytotoxicity of Voriconazole on Cultured Human Corneal Endothelial Cells?  

PubMed Central

The purpose of the present study was to evaluate the toxicity of voriconazole on cultured human corneal endothelial cells (HCECs). HCECs were cultured and exposed to various concentrations of voriconazole (5.0 to 1,000 ?g/ml). Cell viability was measured using a Cell Counting Kit-8 (CCK-8) and live/dead viability/cytotoxicity assays. Cell damage was assessed using phase-contrast microscopy after 24 h of exposure to voriconazole. To analyze the effect of voriconazole on the intercellular barrier, immunolocalization of zonula occludens 1 (ZO1) was performed. A flow cytometric assay was performed to evaluate the apoptotic and necrotic effects of voriconazole on HCECs. Cytotoxicity tests demonstrated the dose-dependent toxic effect of voriconazole on HCECs. Voriconazole concentrations of ?100 ?g/ml led to a significant reduction in cell viability. The morphological characteristics of HCECs also changed in a dose-dependent manner. Increasing concentrations of voriconazole resulted in fading staining for ZO1. Higher concentrations of voriconazole resulted in an increased number of propidium iodide (PI)-positive cells, indicating activation of the proapoptotic pathway. In conclusion, voriconazole may have a dose-dependent toxic effect on cultured HCECs. The results of this study suggest that although voriconazole concentrations of up to 50 ?g/ml do not decrease cell viability, intracameral voriconazole concentrations of ?100 ?g/ml may increase the risk of corneal endothelial damage. PMID:21768517

Han, Sang Beom; Shin, Young Joo; Hyon, Joon Young; Wee, Won Ryang

2011-01-01

134

[Polysaccharides of cell cultures of Silene vulgaris].  

PubMed

Callus and suspension cultures of campion (Silene vulgaris) produced pectin polysaccharides, similar in structure to the polysaccharides of intact plants. The major components of the pectins were D-galacturonic acid, galactose, arabinose, and rhamnose residues. The maximum content of pectins was found in callus. The monosaccharide composition of arabinogalactans isolated from cells and a culture medium of callus cultures were similar, with the ratio between arabinose and galactose of 1: (2.3-6.5) being retained. The arabinogalactans from the cells and culture medium of the suspension cultures also had a similar structure, and the arabinose to galactose ratio was 1: (1.5-1.8). In contrast to the callus cultures, the suspension cultures produced arabinogalactans with an increased content of arabinose residues and a decreased content of galactose residues. The greatest content of arabinogalactan was detected in the culture medium of the suspension cultures. PMID:17345866

Giunter, E A; Ovodov, Iu S

2007-01-01

135

Micropatterned porous membranes for combinatorial cell-based assays.  

PubMed

Here, we describe a protocol for producing micropatterned porous membranes which can be used for combinatorial cell-based assays. We use contact printing to pattern the surface of a porous filter membrane with a thin layer of polydimethylsiloxane (PDMS). This allows the porosity of the filter membrane to be altered at selected locations. Cells can be grown on one side of the filter membrane, while drugs and reagents can be deposited on the porous areas of the other side of the membrane. The reagents can diffuse through the pores of the membrane to the cells. The first part of the protocol describes how to design a stamp and use it to contact print PDMS. The second part describes how to create microprinted membranes for cell-based assays. The method is simple, highly customizable, can be performed at the bench, and can be used to perform combinatorial or time-dependent cell-based assays. PMID:24560509

Vulin, Clément; Evenou, Fanny; Di Meglio, Jean Marc; Hersen, Pascal

2014-01-01

136

Evaluating the effect of assay preparation on the uptake of gold nanoparticles by RAW264.7 cells.  

PubMed

BackgroundCell culture conditions can greatly influence the results of nanoparticle (NP) uptake assays. In this study, 10 nm gold nanoparticles (AuNPs) and RAW 264.7 macrophages were used as a model system, while instrumental neutron activation analysis (NAA) was used as the elemental analysis technique to determine AuNP levels produced by the various culturing conditions. Static plate-based and insert-based culture conditions were compared with a dynamic suspension culture to evaluate the conditions¿ effect on the rate and extent of AuNP uptake.ResultsThe results indicate that a dynamic culturing condition allows for the greatest NP uptake (approximately 3-5 times over the adherent conditions), whereas the plate-based assays have the initial highest rate of NP incorporation.ConclusionsThese data highlight the importance of judiciously choosing the assay conditions prior to evaluating NP uptake. PMID:25424488

Bancos, Simona; Tyner, Katherine M

2014-11-26

137

J Cell Biochem . Author manuscript Optimizing stem cell culture  

E-print Network

cell culture is widely used in basic research for studying stem cell biology, but also owingJ Cell Biochem . Author manuscript Page /1 7 Optimizing stem cell culture Boudewijn Van Der Sanden * Correspondence should be adressed to: Didier Wion Abstract Stem cells always

Paris-Sud XI, Université de

138

A Microwell Cell Culture Platform for the Aggregation of Pancreatic ?-Cells  

PubMed Central

Cell–cell contact between pancreatic ?-cells is important for maintaining survival and normal insulin secretion. Various techniques have been developed to promote cell–cell contact between ?-cells, but a simple yet robust method that affords precise control over three-dimensional (3D) ?-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 ?-cell aggregates of defined sizes from 25 to 210??m in diameter. Using this platform, mouse insulinoma 6 (MIN6) ?-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 ?-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 ?-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 ?-cell aggregates of specific sizes were created, providing a platform from which other biologically relevant questions may be answered. PMID:22320435

Bernard, Abigail B.; Lin, Chien-Chi

2012-01-01

139

Air pollutant production by algal cell cultures  

NASA Technical Reports Server (NTRS)

The production of phytotoxic air pollutants by cultures of Chlorella vulgaris and Euglena gracilis is considered. Algal and plant culture systems, a fumigation system, and ethylene, ethane, cyanide, and nitrogen oxides assays are discussed. Bean, tobacco, mustard green, cantaloupe and wheat plants all showed injury when fumigated with algal gases for 4 hours. Only coleus plants showed any resistance to the gases. It is found that a closed or recycled air effluent system does not produce plant injury from algal air pollutants.

Fong, F.; Funkhouser, E. A.

1982-01-01

140

Culture of Cells from Amphibian Embryos.  

ERIC Educational Resources Information Center

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)

Stanisstreet, Martin

1983-01-01

141

Validation of a miniaturized assay based on IFNg secretion for assessment of specific T cell immunity.  

PubMed

A miniaturized method for detection of antigen induced secretion of IFNg by specific T cells cultured in 384 well plates has been recently reported. In order to confidently apply this assay to clinical investigations for monitoring of specific T cell immunity, an intralaboratory validation study has been undertaken. High reproducibility and linearity of reference curves was demonstrated. Consecutive replicate experiments handled by different operators using broad panels of recall antigens were reproducible when tested on individual biological samples. Kinetics of IFNg secretion with different antigens showed a plateau after 24h culture. Similar trends were observed with secretion of TNFa, GM-CSF and IL17, suggesting that the same kinetics can be applied if other cytokines are tested with this assay. It was demonstrated that frozen-thawed cells can be tested by cell-ELISA and that when PBMC are replaced by whole blood similar reactivity profiles were observed even though cytokine concentration was lower. T cell responses were higher in round bottom than in flat bottom wells, but these plates could not be applied to cell-ELISA as clear plates are not available for scanning. In conclusion, the assay proved flexible, since plates can be frozen at different times during the process, fresh or frozen PBMC and PBMC or whole blood could be used, and robust, since reproducibility was remarkable even when different operators performed the procedures. PMID:20193687

Li Pira, Giusi; Ivaldi, Federico; Moretti, Paolo; Risso, Marco; Tripodi, Gino; Manca, Fabrizio

2010-04-15

142

Automation of 3-dimensional cell culture in arrayed microfluidic devices  

PubMed Central

The increasing interest in studying the interactions between cells and the extracellular matrix (ECM) has created a need for high throughput low cost three-dimensional (3D) culture systems. The recent development of tubeless microfluidics via passive pumping provides a high throughput microchannel culture platform compatible with existing high throughput infrastructures (e.g. automated liquid handlers). Here we build on a previously reported high throughput two-dimensional (2D) system to create a robust automated system for 3D culture. Operational controls including temperature and sample handling have been characterized and automated. Human mammary fibroblasts (HMFs) suspended in type-I collagen are loaded and cultured in microchannel arrays, and used to optimize the system operational parameters. A Peltier cooler maintains the collagen as a liquid at 4°C during cell seeding, followed by polymerization at 37°C. Optimization of this platform is discussed (e.g. controlling collagen contraction, increasing cell viability, preventing the removal of microchannel contents), and 3D distribution of HMFs is examined by fluorescent microscopy. Finally, we validate the platform by automating a previously developed 3D breast carcinoma co-culture assay. The platform allows more efficient 3D culture experiments and lays the foundation for high throughput studies of cell-ECM interactions. PMID:21609700

Montanez-Sauri, Sara I.; Sung, Kyung Eun; Puccinelli, John P.; Pehlke, Carolyn; Beebe, David J.

2011-01-01

143

In vitro screening assay for teratogens using growth inhibition of human embryonic cells  

SciTech Connect

The authors have tested 35 teratogenic and 20 nonteratogenic chemicals or drugs in a short-term, in vitro assay that identifies teratogens by their ability to inhibit growth of an established line of human embryonic palatal mesenchymal cells. Only those chemicals that exhibited a dose-dependent inhibition of growth at concentrations less than 1 mM were classified as inhibitory. An Aroclor-induced rat liver S-9 system was effective in metabolizing cyclophosphamide to its teratogenic form in culture. The authors suggest that this assay, along with the complementary tumor cell-attachment assay of Braun may be useful as a short-term in vitro battery for assessment of the teratogenic potential in environmental agents and to prioritize those chemicals which merit further testing in vivo.

Pratt, R.M.; Willis, W.D.

1985-09-01

144

Embryonic Stem Cells: Isolation, Characterization and Culture  

NASA Astrophysics Data System (ADS)

Embryonic stem cells are pluripotent cells isolated from the mammalian blastocyst. Traditionally, these cells have been derived and cultured with mouse embryonic fibroblast (MEF) supportive layers, which allow their continuous growth in an undifferentiated state. However, for any future industrial or clinical application hESCs should be cultured in reproducible, defined, and xeno-free culture system, where exposure to animal pathogens is prevented. From their derivation in 1998 the methods for culturing hESCs were significantly improved. This chapter wills discuss hESC characterization and the basic methods for their derivation and maintenance.

Amit, Michal; Itskovitz-Eldor, Joseph

145

Three-dimensional culture models of normal and malignant breast epithelial cells  

Microsoft Academic Search

Extracellular matrix is a key regulator of normal homeostasis and tissue phenotype. Important signals are lost when cells are cultured ex vivo on two-dimensional plastic substrata. Many of these crucial microenvironmental cues may be restored using three-dimensional (3D) cultures of laminin-rich extracellular matrix (lrECM). These 3D culture assays allow phenotypic discrimination between nonmalignant and malignant mammary cells, as the former

Genee Y Lee; Paraic A Kenny; Eva H Lee; Mina J Bissell

2007-01-01

146

AMMONIA REMOVAL FROM MAMMALIAN CELL CULTURE MEDIUM  

EPA Science Inventory

Metabolites such as ammonia and lactic formed during mammalian cell culture can frequently be toxic to the cells themselves beyond a threshold concentration of the metabolites. ell culture conducted in the presence of such accumulated metabolites is therefore limited in productiv...

147

Cell-based assays for Parkinson's disease using differentiated human LUHMES cells  

PubMed Central

Aim: Lund human mesencephalic (LUHMES) cells can be differentiated to post-mitotic cells with biochemical, morphological and functional features of dopaminergic (DAergic) neurons. Given the limited scale of primary DAergic neuron culture, we developed differentiated LUHMES cell-based cytotoxicity assays for identifying neuroprotective agents for Parkinson's disease (PD). Methods: LUHMES cells were incubated in a differentiation medium containing cAMP and GDNF for 6 d, and then differentiated cells were treated with MPP+ or infected with baculovirus containing ?-synuclein. Cytotoxicity was determined by measuring intracellular ATP levels and caspase 3/7 activity in the cells. DAergic neuron-specific marker protein and mRNA levels in the cells were analyzed using Western blotting and RT-PCR, respectively. Results: LUHMES cells grew extensive neurites and became post-mitotic neuron-like cells during differentiation period, and three DAergic neuron markers TH, DAT and Nurr1 exhibited different expression profiles. MPP+ dose-dependently reduced ATP levels in the cells with an IC50 value of 65 ?mol/L. MPP+ (80 ?mol/L) significantly increased caspase 3/7 activity in the cells. Both the CDK inhibitor GW8510 and the GSK3? inhibitor SB216763 effectively rescued MPP+-induced reduction of ATP levels with EC50 values of 12 and 205 nmol/L, respectively. Overexpression of ?-synuclein also significantly decreased intracellular ATP levels and increased caspase 3/7 activity in the cells. GW8510 and SB216763 effectively rescued ?-synuclein overexpression-induced reduction of ATP levels, whereas GW8510, but not SB216763, ameliorated ?-synuclein overexpression-induced increase of caspase 3/7 activity. Conclusion: MPP+- and ?-synuclein overexpression-induced cytotoxicity of differentiated LUHMES cells may serve as good alternative systems for identifying neuroprotective compounds for PD. PMID:24989254

Zhang, Xiao-min; Yin, Ming; Zhang, Min-hua

2014-01-01

148

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

PubMed

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?M DCFH-DA and pure phytochemicals with Caco-2 cells for 20min and fluorescence recorded for 90min. 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. PMID:25577125

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

2015-05-15

149

A Real-Time PCR Assay for the Detection of Campylobacter jejuni in Foods after Enrichment Culture  

Microsoft Academic Search

A real-time PCR assay was developed for the quantitative detection of Campylobacter jejuni in foods after enrichment culture. The specificity of the assay for C. jejuni was demonstrated with a diverse range of Campylobacter species, related organisms, and unrelated genera. The assay had a linear range of quantification over six orders of magnitude, and the limit of detection was approximately

Andrew D. Sails; Andrew J. Fox; Frederick J. Bolton; David R. A. Wareing; David L. A. Greenway

2003-01-01

150

Studying cell-cell communication in co-culture  

PubMed Central

Heterotypic and homotypic cellular interactions are essential for biological function, and co-culture models are versatile tools for investigating these cellular interactions in vitro. Physiologically relevant co-culture models have been used to elucidate the effects of cell-cell physical contact and/or secreted factors, as well as the influence of substrate geometry and interaction scale on cell response. Identifying the relative contribution of each cell population to co-culture is often experimentally challenging for these cellular interactions studies. In this issue of Biotechnology Journal, Hamilton et al. [1] report on a hydrogel-based co-culture system, that enables paracrine interactions. A simple and elegant method for enzymatic separation of cell populations post co-culture is introduced, thereby enhancing the ease for post-culture analysis of the effects of co-culture on individual cell populations. PMID:23554248

Bogdanowicz, Danielle R.; Lu, Helen H.

2014-01-01

151

21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.  

Code of Federal Regulations, 2010 CFR

...culture media for human ex vivo tissue and cell culture processing applications. 876...culture media for human ex vivo tissue and cell culture processing applications. (a...culture media for human ex vivo tissue and cell culture processing applications...

2010-04-01

152

21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.  

Code of Federal Regulations, 2011 CFR

...culture media for human ex vivo tissue and cell culture processing applications. 876...culture media for human ex vivo tissue and cell culture processing applications. (a...culture media for human ex vivo tissue and cell culture processing applications...

2011-04-01

153

21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.  

Code of Federal Regulations, 2013 CFR

...culture media for human ex vivo tissue and cell culture processing applications. 876...culture media for human ex vivo tissue and cell culture processing applications. (a...culture media for human ex vivo tissue and cell culture processing applications...

2013-04-01

154

21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.  

Code of Federal Regulations, 2012 CFR

...culture media for human ex vivo tissue and cell culture processing applications. 876...culture media for human ex vivo tissue and cell culture processing applications. (a...culture media for human ex vivo tissue and cell culture processing applications...

2012-04-01

155

21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.  

...culture media for human ex vivo tissue and cell culture processing applications. 876...culture media for human ex vivo tissue and cell culture processing applications. (a...culture media for human ex vivo tissue and cell culture processing applications...

2014-04-01

156

Measuring antimicrobial peptide activity on epithelial surfaces in cell culture  

PubMed Central

To more accurately assess the activity and role of epithelial-cell derived antimicrobial peptides in their native settings, it is essential to perform assays at the surfaces under relevant conditions. In order to carry this out, we utilize 3-dimensional cultures of airway and gingival epithelium, which are grown at an air-liquid interface. Under these conditions, the cultures can be subjected to challenge with a variety of factors known to cause an increase in antimicrobial peptide gene expression. The functional relevance of this induction can then be assessed by quantifying antibacterial activity either directly on the surface of the cells or using the fluid secreted onto the apical surface of the cultures. The relative contribution of the peptides can also be measured by pre-incubation of the secreted fluid with specific inhibitory antibodies. Thus, a relatively inexpensive in vitro model can be used to evaluate the role of antimicrobial peptides in mucosal epithelium. PMID:20094876

Diamond, Gill; Yim, Sunghan; Rigo, Isaura; McMahon, Laura

2009-01-01

157

An easy cell-based microchip assay method for a CYP1A1-mediated drug metabolism using adhesive cells, HepG2  

NASA Astrophysics Data System (ADS)

We have developed a convenient cell-based assay method using a microchip. In the method, adhesive cells, HepG2, were cultured in the conventional culture dish containing glass disks and then the disks covered with the HepG2 were transferred to the microchip for cell assay. Activity of ethoxyresorufin O-deethylation (EROD), which is mainly mediated by cytochrome P4501A1 (CYP1A1), in HepG2 was measured. Treatment of HepG2 with 3-methylcholanthrene, a CYP1A1 inducer, resulted in significant increase in EROD activity.

Kato, Masaru; Yamamoto, Tatsuhiro; Sekimoto, Masashi; Degawa, Masakuni; Toyo'oka, Toshimasa

2009-05-01

158

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

SciTech Connect

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 (/sup 32/Pi). Intra- and extracellular /sup 32/PPi 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 /sup 32/Pi 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.

Prins, A.P.; Kiljan, E.; v.d. Stadt, R.J.; v.d. Korst, J.K.

1986-02-01

159

Method of assay of red cell folate activity and the value of the assay as a test for folate deficiency  

Microsoft Academic Search

A simplified microbiological assay for determining the folate content of red cells is described. As in previously reported methods Lactobacillus casei is used as test organism but two modifications are introduced. First, haemolysis is carried out in water containing 1 g.% of ascorbic acid; secondly, haemolysates are not incubated before the assay. Using this assay, recovery of pteroylglutamic acid added

A. V. Hoffbrand; Beverley F. A. Newcombe; D. L. Mollin

1966-01-01

160

Constructing a High Density Cell Culture System  

NASA Technical Reports Server (NTRS)

An annular culture vessel for growing mammalian cells is constructed in a one piece integral and annular configuration with an open end which is closed by an endcap. The culture vessel is rotatable about a horizontal axis by use of conventional roller systems commonly used in culture laboratories. The end wall of the endcap has tapered access ports to frictionally and sealingly receive the ends of hypodermic syringes. The syringes permit the introduction of fresh nutrient and withdrawal of spent nutrients. The walls are made of conventional polymeric cell culture material and are subjected to neutron bombardment to form minute gas permeable perforations in the walls.

Spaulding, Glenn F. (Inventor)

1996-01-01

161

Emulsions Containing Perfluorocarbon Support Cell Cultures  

NASA Technical Reports Server (NTRS)

Addition of emulsion containing perfluorocarbon liquid to aqueous cell-culture medium increases capacity of medium to support mammalian cells. FC-40 Fluorinert (or equivalent) - increases average density of medium so approximately equal to that of cells. Cells stay suspended in medium without mechanical stirring, which damages them. Increases density enough to prevent cells from setting, and increases viscosity of medium so oxygen bubbled through it and nutrients stirred in with less damage to delicate cells.

Ju, Lu-Kwang; Lee, Jaw Fang; Armiger, William B.

1990-01-01

162

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

EPA Science Inventory

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...

163

Cytokinesis-block micronucleus assay adapted for analyzing genomic instability of human mesenchymal stem cells.  

PubMed

Human mesenchymal stem cells (hMSCs) are multipotent cells used in cell therapy research. One of the problems involving hMSCs is the possibility of genetic instability during in vitro expansion required to obtain a suitable number of cells for clinical applications. The cytokinesis-block micronucleus (CBMN) assay measures genetic instability by analyzing the presence of micronucleus (MN), nucleoplasmic bridges (NPBs), and nuclear buds (NBUDs) in binucleated cells. The present study describes modifications in the CBMN assay methodology to analyze genetic instability in hMSCs isolated from the umbilical vein and in vitro expanded. The best protocol to achieve binucleated hMSCs with preserved cytoplasm was as follows: cytochalasin B concentration (4.0 ?g/mL), use of hypotonic treatment (3 min), and the fixative solution (9 methanol:1 acetic acid). These adaptations were reproduced in three hMSC primary cell cultures and also in XP4PA and A549 cell lines. The frequency of hMSCs treated with mitomycin-C presenting MN was lower than that with other nuclear alterations, indicating that the hMSCs contain mechanisms to avoid a high level of chromosomal breaks. However, a high frequency of cells with NPBs was detected and spontaneous anaphase bridges under normal hMSC in vitro culture were observed. Considering that anaphase bridges are characteristic alterations in tumor cells, the CBMN assay is indicated as an important tool associated with other genetic analyses in order to ensure the safe clinical use of hMSCs in cell therapy. PMID:24328548

Cornélio, Déborah Afonso; Tavares, Joana Cristina Medeiros; Pimentel, Thais Valéria Costa de Andrade; Cavalcanti, Geraldo Barroso; Batistuzzo de Medeiros, Silvia Regina

2014-04-15

164

EVALUATION OF MIXED CELL TYPES AND 5-IODO-2'-DEOXYURIDINE TREATMENT UPON PLAQUE ASSAY TITERS OF HUMAN ENTERIC VIRUSES (JOURNAL VERSION)  

EPA Science Inventory

Four continuous cell lines BGM, L-132, HEL-299, and RD were compared both when cultured separately and as mixtures for use in plaque assay titrations of human Adenovirus 1 and six human enterovirus serotypes. The effect of incubating these cell cultures in media containing IDU (5...

165

Novel Patient Cell-Based HTS Assay for Identification of Small Molecules for a Lysosomal Storage Disease  

PubMed Central

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 opportunity to identify therapeutic small molecules in a disease-cellular environment where potentially disrupted pathways are exposed and available as targets. PMID:22216298

Ribbens, Jameson; Zheng, Wei; Southall, Noel; Hu, Xin; Marugan, Juan J.; Ferrer, Marc; Maegawa, Gustavo H. B.

2011-01-01

166

Action of tumor initiators and promoters in the Syrian hamster embryo cell transformation assay  

SciTech Connect

The Syrian hamster embryo (SHE) cell transformation assay is unique among the rodent fibroblast transformation systems in that it uses normal, diploid cells. Alteration in the control of growth in carcinogen-treated cultures is used to indicate the onset of neoplastic development. An evaluation of the SHE assay for screening carcinogens is reported. Using coded chemicals, the degree of intra- and interlaboratory reproducibility with the system was evaluated. Overall, there was a good qualitative correlation between the carcinogenicity of the chemicals and their ability to induce morphological cell transformation. Unfortunately, the low level of response and lack of good dose-response relationships with certain chemical are still major constraints to the use of this system in routine testing. Further consideration needs to be given to developing procedures that select for, or amplify, expression of the transformed phenotype. 9 refs., 2 figs., 1 tab.

Jones, C.A.; Huberman, E.

1986-06-01

167

Characterization of glucose transport by cultured rabbit kidney proximal convoluted and proximal straight tubule cells  

Microsoft Academic Search

Summary  Rabbit kidney proximal convoluted tubule (RPCT) and proximal straight tubule (RPST) cells were independently isolated and\\u000a cultured. The kinetics of the sodium-dependent glucose transport was characterized by determining the uptake of the glucose\\u000a analog alpha-methylglucopyranoside. Cell culture and assay conditions used in these experiments were based on previous experiments\\u000a conducted on the renal cell line derived from the whole kidney

Pedro L. Del Valle; Anna Trifillis; Charles E. Ruegg; Andrew S. Kane

2002-01-01

168

Assessment of environmental contamination associated with a mammalian cell transformation assay.  

PubMed

To estimate worker exposures to, and environmental contamination from, test chemicals and organic solvents used in an in vitro assay to assess the carcinogenic potential of chemicals, sodium fluorescein, a noncarcinogenic fluorescent material, was dissolved in tissue culture medium used to maintain early passage hamster embryo cells. Personal an environmental samples were taken over a 14-d period. The assay was performed according to standard procedures in a ventilated glove box or laminar flow safety cabinet. Considerably more than 99% of the chemical contamination found was recovered from the interiors of the glove box and hood and from disposable equipment. Contamination outside the containment units (less than 1 microgram) resulted from intralaboratory transport of chemicals, treated cultures, and contaminated equipment. We conclude that the standard operating particles and procedures provided adequate safeguards for personnel and the environment. PMID:7298058

Sansone, E B; Losikoff, A M; Lebherz, W B; Poiley, J A

1981-09-01

169

21 CFR 864.2280 - Cultured animal and human cells.  

Code of Federal Regulations, 2013 CFR

...2013-04-01 false Cultured animal and human cells. 864.2280 Section 864.2280... HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification....

2013-04-01

170

21 CFR 864.2280 - Cultured animal and human cells.  

Code of Federal Regulations, 2012 CFR

...2012-04-01 false Cultured animal and human cells. 864.2280 Section 864.2280... HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification....

2012-04-01

171

21 CFR 864.2280 - Cultured animal and human cells.  

Code of Federal Regulations, 2011 CFR

...2011-04-01 false Cultured animal and human cells. 864.2280 Section 864.2280... HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification....

2011-04-01

172

21 CFR 864.2280 - Cultured animal and human cells.  

Code of Federal Regulations, 2010 CFR

...2010-04-01 false Cultured animal and human cells. 864.2280 Section 864.2280... HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification....

2010-04-01

173

21 CFR 864.2280 - Cultured animal and human cells.  

...2014-04-01 false Cultured animal and human cells. 864.2280 Section 864.2280... HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification....

2014-04-01

174

Cell micropatterning inside a microchannel and assays under a stable concentration gradient.  

PubMed

We describe the use of a microfluidic device to micropattern cells in a microchannel and investigated the behavior of these cells under a concentration gradient. The microfluidic device consisted of 3 parts: a branched channel for generating a stable concentration gradient, a main channel for culturing cells, and 2 side channels that flowed into the main channel. The main channel was coated with a cross-linked albumin that was initially cell-repellent but that could become cell-adherent by electrostatic adsorption of a polycation. A sheath flow stream, which was generated by introducing a polycation solution from the branched channel and a buffer solution from the 2 side channels, was used to change a specific region in the main channel from cell-repellent to cell-adhesive. In this way, cells attached to the central region along the main channel. The remaining surface was subsequently changed to cell-adhesive, thereby facilitating cell migration from a fixed location under a concentration gradient. We demonstrated that with this device, the gradient generator could be used to conduct simultaneous cytotoxic assays with anticancer agents; further, by combining this device with cell micropatterning, migration assays under a concentration gradient of biological factors could be conducted. PMID:20547384

Okuyama, Tomoaki; Yamazoe, Hironori; Seto, Yuki; Suzuki, Hiroaki; Fukuda, Junji

2010-08-01

175

Cancer Phylogenetics from Single-Cell Assays Gregory Pennington  

E-print Network

Cancer Phylogenetics from Single-Cell Assays Gregory Pennington Stanley Shackney Russell Schwartz the Carnegie Mellon University Berkman Faculty Development Fund. #12;Keywords: computational biology, cancer, FISH, phylogeny #12;Abstract In the field of cancer biology, there is currently great interest

176

Hawthorn ( Crataegus monogyna Jacq.) extract exhibits atropine-sensitive activity in a cultured cardiomyocyte assay  

Microsoft Academic Search

Hawthorn (Crataegus spp.) plant extract is used as a herbal alternative medicine for the prevention and treatment of various cardiovascular diseases.\\u000a Recently, it was shown that hawthorn extract preparations caused negative chronotropic effects in a cultured neonatal murine\\u000a cardiomyocyte assay, independent of beta-adrenergic receptor blockade. The aim of this study was to further characterize the\\u000a effect of hawthorn extract to

Satin Salehi; Shannon R. Long; Philip J. Proteau; Theresa M. Filtz

2009-01-01

177

Cell culture processes for monoclonal antibody production  

PubMed Central

Animal cell culture technology has advanced significantly over the last few decades and is now generally considered a reliable, robust and relatively mature technology. A range of biotherapeutics are currently synthesized using cell culture methods in large scale manufacturing facilities that produce products for both commercial use and clinical studies. The robust implementation of this technology requires optimization of a number of variables, including (1) cell lines capable of synthesizing the required molecules at high productivities that ensure low operating cost; (2) culture media and bioreactor culture conditions that achieve both the requisite productivity and meet product quality specifications; (3) appropriate on-line and off-line sensors capable of providing information that enhances process control; and (4) good understanding of culture performance at different scales to ensure smooth scale-up. Successful implementation also requires appropriate strategies for process development, scale-up and process characterization and validation that enable robust operation and ensure compliance with current regulations. This review provides an overview of the state-of-the art technology in key aspects of cell culture, e.g., generation of highly productive cell lines and optimization of cell culture process conditions. We also summarize the current thinking on appropriate process development strategies and process advances that might affect process development. PMID:20622510

Li, Feng; Vijayasankaran, Natarajan; Shen, Amy (Yijuan); Kiss, Robert

2010-01-01

178

Double-layered collagen gel hemisphere for cell invasion assay: successful visualization and quantification of cell invasion activity.  

PubMed

Although various methods for collagen gel-based cell invasion assays have been described, there continues to be a need for a simpler and more objective assay. Here, we describe an easy-to-prepare double-layered collagen gel hemisphere (DL-CGH) system that satisfies these requirements, and we demonstrate the advantages of this new system for visualizing cell movements during invasion. DL-CGH consists of a central core collagen layer surrounded by an outer cover collagen layer. A droplet of collagen I solution (containing cells to be examined) naturally forms a small hemisphere on the bottom of the culture dish. After this central core layer gels, a second droplet is placed atop the first gel, encapsulating it completely. The hemisphere is submerged in the medium and cultured. The invasive activity of cells that infiltrate from the inner to the outer layer can be evaluated optically. Using this in vitro system, we measured the inhibitory effect of E-cadherin expression on cancer cell invasion. DL-CGH also allowed visualization of interactions between invading cancer cells and the stroma. Cancer cells, which lack the proteases required for direct entrance into the three-dimensional collagen matrix, were seen to slip like amoebas through matrix gaps generated by the pericellular proteolytic activity of fibroblasts. [Supplementary materials are available for this article. Go to the publisher's online edition of Cell Communication and Adhesion for the following free supplemental resources: Movies 1-3; 4a and b]. PMID:17957531

Takata, Masahiko; Maniwa, Yoshimasa; Doi, Takefumi; Tanaka, Yugo; Okada, Kenji; Nishio, Wataru; Ohbayashi, Chiho; Yoshimura, Masahiro; Hayashi, Yoshitake; Okita, Yutaka

2007-10-01

179

A Microfluidic Localized, Multiple Cell Culture Array using Vacuum Actuated Cell Seeding: Integrated Anticancer Drug Testing  

PubMed Central

In this study, we introduced a novel and convenient approach to culture multiple cells in localized arrays of microfluidic chambers using one-step vacuum actuation. In one device, we integrated 8 individually addressable regions of culture chambers, each only requiring one simple vacuum operation to seed cells lines. Four cell lines were seeded in designated regions in one device via sequential injection with high purity (99.9%-100%) and cultured for long-term. The on-chip simultaneous culture of HuT 78, Ramos, PC-3 and C166-GFP cells for 48 h was demonstrated with viabilities of 92%+/?2%, 94%+/?4%, 96%+/?2% and 97%+/?2%, respectively. The longest culture period for C166-GFP cells in this study was 168 h with a viability of 96%+/?10%. Cell proliferation in each individual side channel can be tracked. Mass transport between the main channel and side channels was achieved through diffusion and studied using fluorescein solution. The main advantage of this device is the capability to perform multiple cell-based assays on the same device for better comparative studies. After treating cells with staurosporine or anti-human CD95 for 16 h, the apoptotic cell percentage of HuT 78, CCRF-CEM, PC-3 and Ramos cells were 36%+/?3%, 24%+/?4%, 12%+/?2%, 18%+/?4% for staurosporine, and 63%+/?2%, 45%+/?1%, 3%+/?3%, 27%+/?12% for anti-human CD95, respectively. With the advantages of enhanced integration, ease of use and fabrication, and flexibility, this device will be suitable for long-term multiple cell monitoring and cell based assays. PMID:23813077

Gao, Yan; Li, Peng

2013-01-01

180

Advances in cell culture: anchorage dependence.  

PubMed

Anchorage-dependent cells are of great interest for various biotechnological applications. (i) They represent a formidable production means of viruses for vaccination purposes at very large scales (in 1000-6000 l reactors) using microcarriers, and in the last decade many more novel viral vaccines have been developed using this production technology. (ii) With the advent of stem cells and their use/potential use in clinics for cell therapy and regenerative medicine purposes, the development of novel culture devices and technologies for adherent cells has accelerated greatly with a view to the large-scale expansion of these cells. Presently, the really scalable systems--microcarrier/microcarrier-clump cultures using stirred-tank reactors--for the expansion of stem cells are still in their infancy. Only laboratory scale reactors of maximally 2.5 l working volume have been evaluated because thorough knowledge and basic understanding of critical issues with respect to cell expansion while retaining pluripotency and differentiation potential, and the impact of the culture environment on stem cell fate, etc., are still lacking and require further studies. This article gives an overview on critical issues common to all cell culture systems for adherent cells as well as specifics for different types of stem cells in view of small- and large-scale cell expansion and production processes. PMID:25533097

Merten, Otto-Wilhelm

2015-02-01

181

Cultured Porcine Coronary Artery Smooth Muscle Cells  

Microsoft Academic Search

Abstract—Arterial intimal thickening after endothelial injury induced in rodents has proven to be a relatively unreliable model of restenosis for testing clinically useful compounds. The same has been found for cultured rat or rabbit vascular smooth muscle cells (SMCs). To test alternative possibilities, we have studied several differentiation features of porcine coronary artery SMCs, cultured up to the 5th passage

Thomas Christen; Marie-Luce Bochaton-Piallat; Pascal Neuville; Sander Rensen; Mireille Redard; Guillaume van Eys; Giulio Gabbiani

182

Establishment, Characterization, and Toxicological Application of Loggerhead Sea Turtle (Caretta caretta) Primary Skin Fibroblast Cell Cultures.  

PubMed

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 ?M following 72 and 96 h exposures while CYP1A5 induction was detected after 72 h exposure to 0.1-10 ?M 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

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

183

Isolation of mitochondria from tissue culture cells.  

PubMed

The number of mitochondria per cell varies substantially from cell line to cell line. For example, human HeLa cells contain at least twice as many mitochondria as smaller mouse L cells. This protocol starts with a washed cell pellet of 1-2 mL derived from ?10? cells grown in culture. The cells are swollen in a hypotonic buffer and ruptured with a Dounce or Potter-Elvehjem homogenizer using a tight-fitting pestle, and mitochondria are isolated by differential centrifugation. PMID:25275104

Clayton, David A; Shadel, Gerald S

2014-10-01

184

Culture and Manipulation of Embryonic Cells  

PubMed Central

The direct manipulation of embryonic cells is an important tool for addressing key questions in cell and developmental biology. C. elegans is relatively unique among genetic model systems in being amenable to manipulation of embryonic cells. Embryonic cell manipulation has allowed the identification of cell interactions by direct means, and it has been an important technique for dissecting mechanisms by which cell fates are specified, cell divisions are oriented, and morphogenesis is accomplished. Here, we present detailed methods for isolating, manipulating and culturing embryonic cells of C. elegans. PMID:22226523

Edgar, Lois G.; Goldstein, Bob

2012-01-01

185

Cell Culture on MEMS Platforms: A Review  

E-print Network

Microfabricated systems provide an excellent platform for the culture of cells, and are an extremely useful tool for the investigation of cellular responses to various stimuli. Advantages offered over traditional methods ...

Ni, Ming

186

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

PubMed Central

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

Goldstein, G; Guskey, L E

1982-01-01

187

Magnesium variability of lymphocytes from cell culture.  

PubMed

We determined the magnesium content for two different Burkitt's lymphoma cell lines (EW 36 and CA 46) after transfer to new media for 7 consecutive days. Aliquots of the cell culture were washed and the cell pellet was obtained by centrifugation and lysed with distilled water with and without the addition of 0.25% lanthanum oxide. Magnesium was determined by atomic absorption spectrophotometry. The addition of lanthanum permitted the detection of between 8 and 40% more magnesium. The increased magnesium liberated by the addition of lanthanum was calculated as the "bound" magnesium. The results show that the total magnesium is inversely related and the bound magnesium directly related to the age of the cell culture. Thus, there is a decrease of lymphocyte magnesium content and an increase in the percentage of magnesium bound for these two cell lines with increasing age of the cell culture. PMID:4078199

Hosseini, J M; Elin, R J

1985-01-01

188

Responses of the L5178Y tk\\/sup +\\/\\/tk⁻ mouse lymphoma cell forward mutation assay. II. 18 coded chemicals  

Microsoft Academic Search

Eighteen chemicals were tested for their mutagenic potential in the L5178Y tk\\/sup +\\/\\/⁻ mouse lymphoma cell forward mutation assay by the use of procedures based upon those described previously. 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

D. B. McGregor; A. Brown; P. Cattanach; I. Edwards; D. McBride; W. J. Caspary

1988-01-01

189

A New Schizosaccharomyces pombe Chronological Lifespan Assay Reveals that Caloric Restriction Promotes Efficient Cell Cycle Exit and Extends Longevity  

PubMed Central

We describe a new chronological lifespan (CLS) assay for the yeast Schizosaccharomyces pombe. Yeast CLS assays monitor the loss of cell viability in a culture over time, and this new assay shows a continuous decline in viability without detectable regrowth until all cells in the culture are dead. Thus, the survival curve is not altered by the generation of mutants that can grow during the experiments, and one can monitor the entire lifespan of a strain until the number of viable cells has decreased over 106-fold. This CLS assay recapitulates the evolutionarily conserved features of lifespan shortening by over nutrition, lifespan extension by caloric restriction, increased stress resistance of calorically restricted cells and lifespan control by the AKT kinases. Both S. pombe AKT kinase orthologs regulate CLS: loss of sck1+ extended lifespan in over nutrition conditions, loss of sck2+ extended lifespan under both normal and over nutrition conditions, and loss of both genes showed that sck1+ and sck2+ control different longevity pathways. The longest-lived S. pombe cells showed the most efficient cell cycle exit, demonstrating that caloric restriction links these two processes. This new S. pombe CLS assay will provide a valuable tool for aging research. PMID:19409973

Chen, Bo-Ruei; Runge, Kurt W.

2009-01-01

190

Development of a culture system to induce microglia-like cells from haematopoietic cells  

PubMed Central

Aims Microglia are the resident immune cells in the central nervous system, originating from haematopoietic-derived myeloid cells. A microglial cell is a double-edged sword, which has both pro-inflammatory and anti-inflammatory functions. Although understanding the role of microglia in pathological conditions has become increasingly important, histopathology has been the only way to investigate microglia in human diseases. Methods To enable the study of microglial cells in vitro, we here establish a culture system to induce microglia-like cells from haematopoietic cells by coculture with astrocytes. The characteristics of microglia-like cells were analysed by flow cytometry and functional assay. Results We show that triggering receptor expressing on myeloid cells-2-expressing microglia-like cells could be induced from lineage negative cells or monocytes by coculture with astrocytes. Microglia-like cells exhibited lower expression of CD45 and MHC class II than macrophages, a characteristic similar to brain microglia. When introduced into brain slice cultures, these microglia-like cells changed their morphology to a ramified shape on the first day of the culture. Moreover, we demonstrated that microglia-like cells could be induced from human monocytes by coculture with astrocytes. Finally, we showed that interleukin 34 was an important factor in the induction of microglia-like cells from haematopoietic cells in addition to cell–cell contact with astrocytes. Purified microglia-like cells were suitable for further culture and functional analyses. Conclusion Development of in vitro induction system for microglia will further promote the study of human microglial cells under pathological conditions as well as aid in the screening of drugs to target microglial cells. PMID:24016036

Noto, D; Sakuma, H; Takahashi, K; Saika, R; Saga, R; Yamada, M; Yamamura, T; Miyake, S

2014-01-01

191

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

SciTech Connect

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.

Walter, M.N.M. [Institute for Science and Technology in Medicine, Keele University RJAH Orthopaedic Hospital, Oswestry, SY10 7AG (United Kingdom) [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)] [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)] [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)] [School of Life and Health Science, Aston University, Aston Triangle, Birmingham, B4 7EJ (United Kingdom)

2010-04-15

192

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

PubMed

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. PMID:24477854

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

2014-04-01

193

Miniaturized and High-Throughput Assays for Analysis of T-Cell Immunity Specific for Opportunistic Pathogens and HIV  

PubMed Central

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. PMID:24477854

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

2014-01-01

194

Heterotypic cell adhesion assay for the study of cell adhesion inhibition.  

PubMed

CD2 is a cell adhesion molecule that mediates T-cell activation by binding to its ligand CD58 on antigen-presenting cells. Interaction between CD2 and CD58 or leukocyte function-associated antigen-3 (LFA-3) helps to optimize immune recognition facilitating contact between T lymphocytes and antigen-presenting cells. Modulation or inhibition of this interaction has been shown to be therapeutically useful in the treatment of autoimmune diseases. Antibodies and small molecules including peptides have been designed to modulate or disrupt the cell adhesion interactions due to CD2 and CD58. E-rosetting assay is a widely used method applied in the study of the modulation of CD2-CD58 interaction, which is either labor-intensive or radio-hazardous. In this chapter, we describe two methods that are used to study cell adhesion inhibition: (a) E-rosetting Assay and (b) Lymphocyte-epithelial assay. The second method, lymphocyte-epithelial assay, is a rapid and sensitive heterotypic cell adhesion assay for studying cell adhesion inhibition. The method relies on the CD2 expression on the surface of Jurkat cells and the CD58 expression on the surface of Caco-2 cells, which were confirmed by flow cytometry and ELISA studies respectively. This heterotypic cell adhesion assay described typically takes less than 4 h to perform, allows the evaluation of inhibitory activity of peptides/small molecules to modulate CD2-CD58 interaction in real cell system. PMID:21318910

Satyanarayanajois, Seetharama D; Ronald, Sharon; Liu, Jining

2011-01-01

195

Use of a human plaque-forming cell assay to study peripheral blood bursa-equivalent cell activation and excessive suppressor cell activity in humoral immunodeficiency.  

PubMed Central

A plaque assay that detects human mononuclear blood cells producing immunoglobulin (Ig)M antibody to sheep erythrocytes was investigated for its usefulness in studying B-cell activation and regulation in 24 patients with humoral immunodeficiency. Cells from 3 of 15 patients with common variable agammaglobulinemia produced some plaques (range 40--160/10(6) cells; normal range 80--1240/10(6)), but those from the other 12, from all 7 with x-linked agammaglobulinemia and from the 2 with x-linked immunodeficiency with hyper-IgM failed to produce any detectable plaques. In co-cultures of patient and normal cells a very good correlation was seen between results of the plaque assay and an IgM biosynthesis assay in detecting excessive suppressor cell activity. Cells from 7 of 15 common variable agammaglobulinemics, from 3 of 7 x-linked agammaglobulinemics, and from both patients with hyper-IgM caused significant suppression of IgM biosynthesis and(or) plaque formation by normal cells. The observations in the last two groups and discordance for excess suppressor activity in identical twins with common variable agammaglobulinemia suggest that the activity develops secondarily to whatever their primary defects may be. Culturing non-T cells from common variable agammaglobulinemics exhibiting excessive suppressor cell activity with normal T cells resulted in plaque formation in four of five patients so studied; in all five the suppressor activity was found in the T-cell population. The availability of a plaque assay for the study of blood cells from immunodeficient patients provides a new probe to examine the cellular nature of such defects. PMID:376549

Herrod, H G; Buckley, R H

1979-01-01

196

Assay for characterizing the recovery of vertebrate cells for adhesion measurements by single-cell force spectroscopy.  

PubMed

Single-cell force spectroscopy (SCFS) is becoming a widely used method to quantify the adhesion of a living cell to a substrate, another cell or tissue. The high sensitivity of SCFS permits determining the contributions of individual cell adhesion molecules (CAMs) to the adhesion force of an entire cell. However, to prepare adherent cells for SCFS, they must first be detached from tissue-culture flasks or plates. EDTA and trypsin are often applied for this purpose. Because cellular properties can be affected by this treatment, cells need to recover before being further characterized by SCFS. Here we introduce atomic force microscopy (AFM)-based SCFS to measure the mechanical and adhesive properties of HeLa cells and mouse embryonic kidney fibroblasts while they are recovering after detachment from tissue-culture. We find that mechanical and adhesive properties of both cell lines recover quickly (<10 min) after detachment using EDTA, while trypsin-detached fibroblasts require >60 min to fully recover. Our assay introduced to characterize the recovery of mammalian cells after detachment can in future be used to estimate the recovery behavior of other adherent cell types. PMID:24928443

Schubert, Rajib; Strohmeyer, Nico; Bharadwaj, Mitasha; Ramanathan, Subramanian P; Krieg, Michael; Friedrichs, Jens; Franz, Clemens M; Muller, Daniel J

2014-10-01

197

Isolation and culture of pulmonary endothelial cells.  

PubMed

Methods for isolation, identification and culture of pulmonary endothelial cells are now routine. In the past, methods of isolation have used proteolytic enzymes to detach cells; thereafter, traditional methods for cell passaging have used trypsin/EDTA mixtures. Cells isolated and passaged using proteolytic enzymes have been useful in establishing the field and in verifying certain endothelial properties. However, there is a growing awareness of the role of endothelial cells in processing vasoactive substances, in responding to hormones and other agonists and in cell-cell interactions with other cell types of the vascular wall, with blood cells and with cellular products. Consequently, a new requirement has arisen for cells in vitro that maintain the differentiated properties of their counterparts in vivo. The deleterious effects of trypsin and other proteolytic enzymes commonly used in cell culture on surface structures of endothelial cells such as enzymes, receptors and junctional proteins, as well as on extracellular layers such as the glycocalyx or "endothelial fuzz," have led to the development of methods that avoid use of proteolytic enzymes at both the isolation step and during subsequent subculture. This chapter describes traditional methods for isolating pulmonary endothelial cells but emphasizes newer approaches using mechanical harvest and scale-up using microcarriers. The new methods allow maintenance of long-term, large-scale cultures of cells that retain the full complement of surface properties and that maintain the cobblestone monolayer morphology and differentiated functional properties. Methods for identification of isolated cells are therefore also considered as methods for validation of cultures during their in vitro lifespan. PMID:6090112

Ryan, U S

1984-06-01

198

Proximity Ligation Assay for High-content Profiling of Cell Signaling Pathways on a Microfluidic Chip*  

PubMed Central

Here, we present the full integration of a proximity ligation assay (PLA) on a microfluidic chip for systematic cell signaling studies. PLA is an in situ technology for the detection of protein interaction, post-translational modification, concentration, and cellular location with single-molecule resolution. Analytical performance advances on chip are achieved, including full automation of the biochemical PLA steps, target multiplexing, and reduction of antibody consumption by 2 orders of magnitude relative to standard procedures. In combination with a microfluidic cell-culturing platform, this technology allows one to gain control over 128 cell culture microenvironments. We demonstrate the use of the combined cell culture and protein analytic assay on chip by characterizing the Akt signaling pathway upon PDGF stimulation. Signal transduction is detected by monitoring the phosphorylation states of Akt, GSK-3?, p70S6K, S6, Erk1/2, and mTOR and the cellular location of FoxO3a in parallel with the PLA. Single-cell PLA results revealed for Akt and direct targets of Akt a maximum activation time of 4 to 8 min upon PDGF stimulation. Activation times for phosphorylation events downward in the Akt signaling pathway including the phosphorylation of S6, p70S6K, and mTOR are delayed by 8 to 10 min or exhibit a response time of at least 1 h. Quantitative confirmation of the Akt phosphorylation signal was determined with the help of a mouse embryonic fibroblast cell line deficient for rictor. In sum, this work with a miniaturized PLA chip establishes a biotechnological tool for general cell signaling studies and their dynamics relevant for a broad range of biological inquiry. PMID:24072685

Blazek, Matthias; Betz, Charles; Hall, Michael Nip; Reth, Michael; Zengerle, Roland; Meier, Matthias

2013-01-01

199

Barnacle in vitro assays for biologically active substances: Toxicity and Settlement inhibition assays using mass cultured Balanus amphitrite amphitrite darwin  

Microsoft Academic Search

The development of non?toxic or non?polluting antifouling additives that can be formulated in practical coatings requires assays involving target organisms. Assays that test both for the effective and toxic concentrations of active compounds are useful. It is also desirable if the assay can provide information regarding the performance that can be expected if the compounds are incorporated into different matrices.

D. Rittschof; A. S. Clare; D. J. Gerhart; Sister Avelin Mary; J. Bonaventura

1992-01-01

200

Quasi-spherical microwells on superhydrophobic substrates for long term culture of multicellular spheroids and high throughput assays.  

PubMed

Multicellular tumour spheroids closely recapitulate the physiological environment of tumour tissues. However, their implementation in drug screening assays remains limited due to the technological challenges of forming large numbers of high quality spheroids in platforms compatible with high throughput screening. A simple bench-top microfabrication strategy is demonstrated here based on the principle of ice lithography carried out on superhydrophobic substrates to fabricate quasi-spherical microwells (spheriwells). The microwells shapes and dimensions are directly controlled by the hydrophobicity of the substrate and the volume of the water droplets. The prepared concave microwells enable the formation of dense and homogeneous multicellular tumour spheroids. Spheroids formed within spheriwells are trapped within the microwells, which eliminate loss during media manipulation and facilitate long-term on-chip culture. Morphological and phenotypical changes associated with the growth of MCF-7 adenocarcinoma cells in spheriwells were characterised using imaging flow cytometry and revealed the appearance of heterogeneous populations with loss of E-Cadherin expression. The compatibility of the spheriwells with an on-chip MTT assay is demonstrated. The very unusual shape of the spheriwells, prepared using materials and methods routinely used in most research laboratories, provides a straightforward and scalable platform to prepare high quality multicellular tumour spheroids compatible with high throughput biological screening assays. PMID:24797879

Liu, Tianqing; Winter, Marnie; Thierry, Benjamin

2014-07-01

201

Paraquat induces apoptosis of cultured rat cortical cells.  

PubMed

Paraquat (PQ; 1,1'-dimethyl-4,4'-bipyridinium dichloride) is widely used as a universal herbicide. Although systemic treatment with PQ gives rise to the highest level of the herbicide in the cerebral cortex, our knowledge of its effects in this brain region is very limited. We took advantage of rat cortical cell cultures to analyze how PQ affects cortical neurons. Lactate dehydrogenase (LDH) assay and propidium iodide (PI) staining showed that PQ was cytotoxic to cortical neurons with an IC50 on the third day after treatment of approximately 10 microM. PQ-treated cells had shrunken soma with condensed nuclei and disintegrated dendrites, typical signs of apoptosis. Immunocytochemistry of 8-day in vitro (DIV) cells one day after PQ treatment with anti-phospho-H2AX antibody showed that the average number of punctae per nucleus had increased several-fold, indicating substantial DNA fragmentation. Furthermore, double-staining of 7.5 DIV cultures (50 microM PQ) with PI and an antibody against annexin V (AN), an impermeable plasma protein which specifically binds to phosphatidylserine (PS), showed that the percentages of AN(+)/PI(-) cells had also increased several-fold, pointing to considerable movement of PS from the inner to the outer leaflet of the plasma membrane. Taken together, our data indicate that PQ induces apoptosis in cortical cell cultures. PMID:15055535

Kim, Sung Jin; Kim, Jang Eok; Moon, Il Soo

2004-02-29

202

Chromatin reorganization and endogenous auxin\\/cytokinin dynamic activity during somatic embryogenesis of cultured cotton cell  

Microsoft Academic Search

We conducted a systematic assessment and comparative study on the biochemical and cellular characteristics of cultured cotton\\u000a cells during the entire process of somatic embryogenesis (SE). All staged cultures were widely investigated in this assay.\\u000a Cell and tissue ectogenesis manipulation combined with flow cytometry (FCM) was employed to cellular study during the whole\\u000a totipotency process of dedifferentiation and redifferentiation. We

Fanchang Zeng; Xianlong Zhang; Shuangxia Jin; Lei Cheng; Shaoguang Liang; Lisong Hu; Xiaoping Guo; Yichun Nie; Jinglin Cao

2007-01-01

203

Human cell culture in a space bioreactor  

NASA Technical Reports Server (NTRS)

Microgravity offers new ways of handling fluids, gases, and growing mammalian cells in efficient suspension cultures. In 1976 bioreactor engineers designed a system using a cylindrical reactor vessel in which the cells and medium are slowly mixed. The reaction chamber is interchangeable and can be used for several types of cell cultures. NASA has methodically developed unique suspension type cell and recovery apparatus culture systems for bioprocess technology experiments and production of biological products in microgravity. The first Space Bioreactor was designed for microprocessor control, no gaseous headspace, circulation and resupply of culture medium, and slow mixing in very low shear regimes. Various ground based bioreactors are being used to test reactor vessel design, on-line sensors, effects of shear, nutrient supply, and waste removal from continuous culture of human cells attached to microcarriers. The small Bioreactor is being constructed for flight experiments in the Shuttle Middeck to verify systems operation under microgravity conditions and to measure the efficiencies of mass transport, gas transfer, oxygen consumption and control of low shear stress on cells.

Morrison, Dennis R.

1988-01-01

204

Transferring isolated mitochondria into tissue culture cells  

PubMed Central

We have developed a new method for introducing large numbers of isolated mitochondria into tissue culture cells. Direct microinjection of mitochondria into typical mammalian cells has been found to be impractical due to the large size of mitochondria relative to microinjection needles. To circumvent this problem, we inject isolated mitochondria through appropriately sized microinjection needles into rodent oocytes or single-cell embryos, which are much larger than tissue culture cells, and then withdraw a ‘mitocytoplast’ cell fragment containing the injected mitochondria using a modified holding needle. These mitocytoplasts are then fused to recipient cells through viral-mediated membrane fusion and the injected mitochondria are transferred into the cytoplasm of the tissue culture cell. Since mouse oocytes contain large numbers of mouse mitochondria that repopulate recipient mouse cells along with the injected mitochondria, we used either gerbil single-cell embryos or rat oocytes to package injected mouse mitochondria. We found that the gerbil mitochondrial DNA (mtDNA) is not maintained in recipient rho0 mouse cells and that rat mtDNA initially replicated but was soon completely replaced by the injected mouse mtDNA, and so with both procedures mouse cells homoplasmic for the mouse mtDNA in the injected mitochondria were obtained. PMID:22753025

Yang, Yi-Wei; Koob, Michael D.

2012-01-01

205

Calculator programmed assistance in making cell cultures  

Microsoft Academic Search

Summary A calculator program and instructions are presented for facilitating the preparation of single cell suspensions to be used in cell culture systems. The program has been written for the Texas Instruments Inc. TI-59 programmable calculator with printer. The instructions provided are intended for the novice with no previous programming experience and only a basic understanding of the calculator operation.

J. C. Bullaro

1980-01-01

206

Isolating phagosomes from tissue culture cells.  

PubMed

Phagocytosis is the process by which receptors at the plasma membrane are used to engulf a particle such as a bacterium, parasite, or dead cell. Phagosomes can be isolated from tissue culture cells by various centrifugation methods, including the use of differential density gradients or sucrose step gradients, but these methods are time-consuming or otherwise difficult. We describe here a protocol that avoids centrifugation and relies instead on the uptake of magnetic beads to rapidly isolate the phagosomal compartment from tissue culture cells. PMID:25447278

Pryor, Paul R; Rofe, Adam P

2014-12-01

207

Circadian gene expression in cultured cells.  

PubMed

In mammals, circadian oscillators not only exist in specialized neurons of the suprachiasmatic nucleus, but in almost all peripheral cell types. These oscillators are operative even in established fibroblast cell lines, such as Rat-1 cells or NIH3T3 cells, and in primary fibroblasts from mouse embryos or adult animals. This can be demonstrated by treating such cells for a short time period with high concentrations of serum or chemicals that activate a large number of known signaling pathways. The possibility of studying circadian rhythms in cultured cells should facilitate the biochemical and genetic dissection of the circadian clockwork and should promote the discovery of new clock components. PMID:15817311

Nagoshi, Emi; Brown, Steven A; Dibner, Charna; Kornmann, Benoît; Schibler, Ueli

2005-01-01

208

The Millipore filter assay technique for measuring tritiated thymidine incorporation into DNA in leucocyte cultures  

PubMed Central

In this paper we present in detail the very rapid and sensitive Millipore filter assay technique for measuring tritiated thymidine incorporation during semiconservative DNA synthesis in human peripheral blood leucocyte cultures. Leucocytes which have been incubated with tritiated thymidine are trapped on a Millipore filter which is then suitably washed and dried. The filter is then immersed in scintillation fluid in a double-vial apparatus designed to ensure a reproducible counting geometry. Alternatively, the filter and its radioactive contents can be combusted to tritiated water, the radioactivity of which is then determined in a homogeneous counting system. The parameters related to the Millipore filter assay technique are presented, and its present uses and general applicability discussed. PMID:5085245

Robbins, J. H.; Gart, J. J.; Levis, W. R.; Burk, P. G.

1972-01-01

209

Cell culture experiments planned for the space bioreactor  

NASA Technical Reports Server (NTRS)

Culturing of cells in a pilot-scale bioreactor remains to be done in microgravity. An approach is presented based on several studies of cell culture systems. Previous and current cell culture research in microgravity which is specifically directed towards development of a space bioprocess is described. Cell culture experiments planned for a microgravity sciences mission are described in abstract form.

Morrison, Dennis R.; Cross, John H.

1987-01-01

210

White Blood Cell-Based Detection of Asymptomatic Scrapie Infection by Ex Vivo Assays  

PubMed Central

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. PMID:25122456

Halliez, Sophie; Jaumain, Emilie; Huor, Alvina; Douet, Jean-Yves; Lugan, Séverine; Cassard, Hervé; Lacroux, Caroline; Béringue, Vincent; Andréoletti, Olivier; Vilette, Didier

2014-01-01

211

Characteristics of cardiac cell cultures derived from human myocardial explants.  

PubMed

Primary cell cultures derived from human myocardial explants were obtained and characterized. The explant cultures contained cardiac stem cells (c-kit(+); ? 4%), microvascular cells (endothelial cells and pericytes), fibroblasts, and myofibroblasts. It was demonstrated that culturing of cardiac cells in cardiospheres did not promote enrichment of the cell culture with stem cells. MACS-sorted c-kit(+) cells from the explant culture were characterized by limited proliferative capacity and were capable of cardiomyogenic differentiation. The presence of microvascular cells determined general angiogenic potential of the culture. PMID:24319709

Pavlova, S V; Perovskii, P P; Chepeleva, E V; Malakhova, A A; Dement'eva, E V; Pokushalov, E A; Sukhikh, G T; Zakiyan, S M

2013-11-01

212

Development of an ELISA assay for the quantification of soluble huntingtin in human blood cells  

PubMed Central

Background Huntington’s disease (HD) is a monogenic disorder caused by an aberrant expansion of CAG repeats in the huntingtin gene (HTT). Pathogenesis is associated with expression of the mutant (mHTT) protein in the CNS, with its levels most likely related to disease progression and symptom severity. Since non-invasive methods to quantify HTT in the CNS do not exist, measuring amount of soluble HTT in peripheral cells represents an important step in development of disease-modifying interventions in HD. Results An ELISA assay using commercially available antibodies was developed to quantify HTT levels in complex matrices like mammalian cell cultures lysates and human samples. The immunoassay was optimized using a recombinant full-length HTT protein, and validated both on wild-type and mutant HTT species. The ability of the assay to detect significant variations of soluble HTT levels was evaluated using an HSP90 inhibitor that is known to enhance HTT degradation. Once optimized, the bioassay was applied to peripheral blood mononuclear cells (PBMCs) from HD patients, demonstrating good potential in tracking the disease course. Conclusions The method described here represents a validated, simple and rapid bio-molecular assay to evaluate soluble HTT levels in blood cells as useful tool in disease and pharmacodynamic marker identification for observational and clinical trials. PMID:24274906

2013-01-01

213

Fluorescent Cell Counting as an Assay Method for Respiratory Syncytial Virus  

PubMed Central

The fluorescent cell-counting technique was applied to the enumeration of cell-infecting units of respiratory syncytial (RS) virus in human fetal diploid (HFD) cover-slip cell cultures; it was a sensitive, precise, and rapid assay method. Approximately 2 hr was required for maximal adsorption of RS virus to HFD cell monolayers. However, about 15% of the infectious virus in the inoculum remained unadsorbed; this percentage was not significantly reduced even when the adsorption period was extended to 5 hr. A linear relationship between virus concentration and the number of fluorescent cells existed over a range of 1.2 log10 units. Variation of the mean of replicate determinations in a single experiment was approximately 7.5%. The distribution of single infected HFD cells on cover-slip cell cultures corresponded with the calculated frequencies of the Poisson distribution. The Chi square test for the extent of fit was calculated for several experiments, and the value of P was never less than 0.5. The addition of immune serum after virus adsorption effectively inhibited the development of detectable levels of viral antigen in secondarily infected cells. PMID:4918238

Schieble, Jack H.; Kase, Alice; Lennette, Edwin H.

1967-01-01

214

Fluorescent cell counting as an assay method for respiratory syncytial virus.  

PubMed

The fluorescent cell-counting technique was applied to the enumeration of cell-infecting units of respiratory syncytial (RS) virus in human fetal diploid (HFD) cover-slip cell cultures; it was a sensitive, precise, and rapid assay method. Approximately 2 hr was required for maximal adsorption of RS virus to HFD cell monolayers. However, about 15% of the infectious virus in the inoculum remained unadsorbed; this percentage was not significantly reduced even when the adsorption period was extended to 5 hr. A linear relationship between virus concentration and the number of fluorescent cells existed over a range of 1.2 log(10) units. Variation of the mean of replicate determinations in a single experiment was approximately 7.5%. The distribution of single infected HFD cells on cover-slip cell cultures corresponded with the calculated frequencies of the Poisson distribution. The Chi square test for the extent of fit was calculated for several experiments, and the value of P was never less than 0.5. The addition of immune serum after virus adsorption effectively inhibited the development of detectable levels of viral antigen in secondarily infected cells. PMID:4918238

Schieble, J H; Kase, A; Lennette, E H

1967-06-01

215

326 assessment of porcine-induced pluripotent stem cells by in vivo assays.  

PubMed

Porcine-induced pluripotent stem cells (piPSC) have been established since 2009, but only 1 report demonstrated contribution to germline chimeras. One well-established in vivo pluripotency assay is the teratoma assay, which has recently been questioned due to the lack of standardized guidelines. In the present study we have characterised GFP-tagged in vitro and in vivo tetracycline-inducible piPSC [porcine MYC, SOX2, KLF4 (pOMSK)] and their capacity to form teratomas. We injected 1.5 million cells in 250µL of PBS subcutaneous into NOD/SCID mice and followed them up to 6 weeks. The teratomas were analysed by immunohistochemistry for the 3 germlayer markers ?3 tubulin, ? fetoprotein, and smooth muscle actin. We not only found our teratomas positive for these markers, but also co-positive for GFP, clearly showing that the teratoma was made from porcine cells, which was not sufficiently proven in former studies. Our H&E staining revealed the following structures: cuboidal ephitelium, thyroid-like structure, renal corpuscle, and steroid producing cells. We continued to test the capacity of our venus iPS cells to contribute to in vitro chimeras. To achieve this we used a micromanipulator to inject 15 cells into Day 5 parthenotes, and subsequently cultured them in PZM3 with 10% FCS, cultured with or without doxycycline. These in vitro chimeras were followed until Day 7 in Nikons Biostation IM and used for differential staining. In all groups we saw good survival, hatching, and maintenance of GFP, indicating integration of these cells in our in vitro assay. We only found differences between survivals of the cell lines in the group cultured with doxycycline. Finally, in order to assess if the naïve type venus iPS cells could possibly be a truly naïve piPSC, we tested their capacity to form in vivo germline chimeras. This was tested by injecting 15 piPSC into Day 4 to 5 in vivo embryos. The injected embryos were transferred into 5 surrogate mothers, 3 of them were fed doxycycline before the transfer and 5 days after, and the last 2 recipient sows were not fed doxycycline. The pregnancies were terminated at Day 32 and the embryos were examined for fluorescence and the GFP transgene by PCR. In summary, it appears that both naïve type and primed type venus iPS cells are still strongly dependent on the pOMSK transgene expression, and the ultimate proof for pluripotency, the chimera production, seems to be not achievable under the condition we have chosen. PMID:25472374

Secher, J; Freude, K; Petkov, S; Ceylan, A; Schmidt, M; Hyttel, P

2014-12-01

216

Genotoxicity of complex mixtures: CHO cell mutagenicity assay  

SciTech Connect

A Chinese hamster ovary (CHO) mammalian cell assay was used to evaluate the genotoxicity of complex mixtures (synthetic fuels). The genotoxicity (mutagenic potency) of the mixtures increased as the temperature of their boiling range increased. Most of the genotoxicity in the 750/sup 0/F+ boiling-range materials was associated with the neutral polycyclic aromatic hydrocarbon (PAH) fractions. Chemical analysis data indicate that the PAH fractions of high-boiling coal liquids contain a number of known chemical carcinogens, including five- and six-ring polyaromatics (e.g., benzo(a)pyrene) as well as four- and five-ring alkyl-substituted PAH (e.g., methylchrysene and dimethylbenzanthracenes); concentrations are a function of boiling point (bp). In vitro genotoxicity was also detected in fractions of nitrogen-containing polyaromatic compounds, as well as in those with aliphatics of hydroxy-containing PAH. Mutagenic activity of some fractions was detectable in the CHO assay in the absence of an exogenous metabolic activation system; in some instances, addition of exogenous enzymes and cofactors inhibited expression of the direct-acting mutagenic potential of the fraction. These data indicate that the organic matrix of the chemical fraction determines whether, and to what degree, various mutagens are expressed in the CHO assay. Therefore, the results of biological assays of these mixtures must be correlated with chemical analyses for proper interpretation of these data. 29 references, 16 figures, 4 tables.

Frazier, M.E.; Samuel, J.E.

1985-02-01

217

Enhanced growth medium and method for culturing human mammary epithelial cells  

DOEpatents

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.

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

218

Canine coronavirus induces apoptosis in cultured cells  

Microsoft Academic Search

Canine coronavirus (CCoV) is widespread in dogs in several countries and causes mild enteric illness evolving to severe enteritis in young pups.In in vitro cultures canine coronaviruses generally induce extensive cell death, however nature of the events leading to cell death remains largely unknown.We analysed the induction of cytopathic effect by CCoV in a canine fibrosarcoma cell line (A-72) in

A. Ruggieri; L. Di Trani; I. Gatto; M. Franco; E. Vignolo; B. Bedini; G. Elia; C. Buonavoglia

2007-01-01

219

Fetal calf sera can distort cell-based luminescent proteasome assays through heat-resistant chymotrypsin-like activity.  

PubMed

Luminescence-based proteasome activity assays use specific substrates that are supposed to be cleaved by cellular proteasome activity leading to luciferase substrates. Usually, control wells containing cell culture medium supplemented with antibiotics and fetal calf serum are used as background. Using the Proteasome-Glo chymotrypsin-like cell-based assay from Promega, we show here that fetal calf sera from different manufacturers contain heat-resistant, bortezomib-inhibitable, chymotrypsin-like activities that can interfere with proteasome activity assays. These data strongly recommend the use of pure phosphate-buffered saline (PBS) or serum-free medium during proteasome activity assays to diminish background luminescence and, thus, to obtain reliable results. PMID:25447494

Dettmer, Susan; Theile, Dirk; Seckinger, Anja; Burhenne, Jürgen; Weiss, Johanna

2015-02-15

220

Establishment of a New Cell-Based Assay To Measure the Activity of Sweeteners in Fluorescent Food Extracts  

PubMed Central

Taste receptors have been defined at the molecular level in the past decade, and cell-based assays have been developed using cultured cells heterologously expressing these receptors. The most popular approach to detecting the cellular response to a tastant is to measure changes in intracellular Ca2+ concentration using Ca2+-sensitive fluorescent dyes. However, this method cannot be applied to food-derived samples that contain fluorescent substances. To establish an assay system that would be applicable to fluorescent samples, we tested the use of Ca2+-sensitive photoproteins, such as aequorin and mitochondrial clytin-II, as Ca2+ indicators in a human sweet taste receptor assay. Using these systems, we successfully detected receptor activation in response to sweetener, even when fluorescent compounds coexisted. This luminescence-based assay will be a powerful tool to objectively evaluate the sweetness of food-derived samples even at an industry level. PMID:21981007

2011-01-01

221

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

PubMed

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. PMID:20213215

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

2010-06-01

222

Localization of glycosidases in the wall of living cells from cultured Convolvulus arvensis tissue  

Microsoft Academic Search

Nine glycosidase activities were detected in isolated cell walls of cultured Convolvulus arvensis L. cells. Seven were also found in the cytoplasmic fraction. Optimal pH values were all in the acid region. The in vivo localization of some of these glycosidases was studied by assaying whole cells. Suspended cells hydrolysed the externally supplied substrates for a-galactosidase (EC 3.2.1.22), ß-galactosidase (EC

Han Pierrot; John E. van Wielink

1977-01-01

223

Electrophoretic mobilities of cultured human embryonic kidney cells in various buffers  

NASA Technical Reports Server (NTRS)

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.

1985-01-01

224

Detection of Nonhemagglutinating Influenza A(H3) Viruses by Enzyme-Linked Immunosorbent Assay in Quantitative Influenza Virus Culture  

PubMed Central

To assess the efficacy of novel antiviral drugs against influenza virus in clinical trials, it is necessary to quantify infectious virus titers in respiratory tract samples from patients. Typically, this is achieved by inoculating virus-susceptible cells with serial dilutions of clinical specimens and detecting the production of progeny virus by hemagglutination, since influenza viruses generally have the capacity to bind and agglutinate erythrocytes of various species through their hemagglutinin (HA). This readout method is no longer adequate, since an increasing number of currently circulating influenza A virus H3 subtype (A[H3]) viruses display a reduced capacity to agglutinate erythrocytes. Here, we report the magnitude of this problem by analyzing the frequency of HA-deficient A(H3) viruses detected in The Netherlands from 1999 to 2012. Furthermore, we report the development and validation of an alternative method for monitoring the production of progeny influenza virus in quantitative virus cultures, which is independent of the capacity to agglutinate erythrocytes. This method is based on the detection of viral nucleoprotein (NP) in virus culture plates by enzyme-linked immunosorbent assay (ELISA), and it produced results similar to those of the hemagglutination assay using strains with good HA activity, including A/Brisbane/059/07 (H1N1), A/Victoria/210/09 (H3N2), other seasonal A(H1N1), A(H1N1)pdm09, and the majority of A(H3) virus strains isolated in 2009. In contrast, many A(H3) viruses that have circulated since 2010 failed to display HA activity, and infectious virus titers were determined only by detecting NP. The virus culture ELISA described here will enable efficacy testing of new antiviral compounds in clinical trials during seasons in which nonhemagglutinating influenza A viruses circulate. PMID:24622097

Els, C.; Sprong, L.; van Beek, R.; van der Vries, E.; Osterhaus, A. D. M. E.; Rimmelzwaan, G. F.

2014-01-01

225

Three-Dimensional Cultures of Mouse Mammary Epithelial Cells  

PubMed Central

The mammary gland is an ideal “model organism” for studying tissue specificity and gene expression in mammals: it is one of the few organs that develop after birth and it undergoes multiple cycles of growth, differentiation and regression during the animal’s lifetime in preparation for the important function of lactation. The basic “functional differentiation” unit in the gland is the mammary acinus made up of a layer of polarized epithelial cells specialized for milk production surrounded by myoepithelial contractile cells, and the two-layered structure is surrounded by basement membrane. Much knowledge about the regulation of mammary gland development has been acquired from studying the physiology of the gland and of lactation in rodents. Culture studies, however, were hampered by the inability to maintain functional differentiation on conventional tissue culture plastic. We now know that the microenvironment, including the extracellular matrix and tissue architecture, plays a crucial role in directing functional differentiation of organs. Thus, in order for culture systems to be effective experimental models, they need to recapitulate the basic unit of differentiated function in the tissue or organ and to maintain its three-dimensional (3D) structure. Mouse mammary culture models evolved from basic monolayers of cells to an array of complex 3D systems that observe the importance of the microenvironment in dictating proper tissue function and structure. In this chapter, we focus on how 3D mouse mammary epithelial cultures have enabled investigators to gain a better understanding of the organization, development and function of the acinus, and to identify key molecular, structural, and mechanical cues important for maintaining mammary function and architecture. The accompanying chapter of Vidi et al. describes 3D models developed for human cells. Here, we describe how mouse primary epithelial cells and cell lines—essentially those we use in our laboratory—are cultured in relevant 3D microenvironments. We focus on the design of functional assays that enable us to understand the intricate signaling events underlying mammary gland biology, and address the advantages and limitations of the different culture settings. Finally we also discuss how advances in bioengineering tools may help towards the ultimate goal of building tissues and organs in culture for basic research and clinical studies. PMID:23097110

Mroue, Rana; Bissell, Mina J.

2013-01-01

226

Genotoxicity studies of methyl isocyanate in Salmonella, Drosophila, and cultured Chinese hamster ovary cells  

SciTech Connect

The genotoxic effects of methyl isocyanate (MIC) were investigated using four short-term tests: the Salmonella reversion assay (Ames test), the Drosophila sex-linked recessive lethal assay, and the sister chromatic exchange (SCE) and chromosomal aberration assays in cultured Chinese hamster ovary (CHO) cells. No evidence was found for the induction of mutations in either Salmonella or Drosophila. MIC did, however, induce SCEs and chromosomal aberrations in CHO cells both in the presence and absence of Aroclor-induced rat liver S-9.

Mason, J.M.; Zeiger, E.; Haworth, S.; Ivett, J.; Valencia, R.

1987-01-01

227

Fluorous solvent for cell culture  

Microsoft Academic Search

Incubation of mouse melanoma B16 cells in fluorous solvents with low boiling point such as perfluoromethylcyclohexane, 1,1,1,3,3,3-hexafluoro-2-propanol, ethylpentafluoropropionate resulted in cell death. However, cells lived up to 2 days in fluorous alcohols such as 2,2,3,3,4,4,5,5-octafluoro-1-pentanol and 3,3,4,4,5,5,6,6,6-nonafluoro-1-hexanol with relatively higher fluorine content. Remarkably, cells survived deprived of nutrition up to 4 days when incubated in 2,2,3,3,4,4,5,5,6,6,6-undecafluoro-1-hexanol or in 2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptanol that

Maria Carmelita Z. Kasuya; Xiaonan Wen; Kenichi Hatanaka; Kageyasu Akashi

2011-01-01

228

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.  

PubMed

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 ?g ml?¹ chloramphenicol, 100 ?g 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

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

2010-11-01

229

Cell Culture on MEMS Platforms: A Review  

PubMed Central

Microfabricated systems provide an excellent platform for the culture of cells, and are an extremely useful tool for the investigation of cellular responses to various stimuli. Advantages offered over traditional methods include cost-effectiveness, controllability, low volume, high resolution, and sensitivity. Both biocompatible and bio-incompatible materials have been developed for use in these applications. Biocompatible materials such as PMMA or PLGA can be used directly for cell culture. However, for bio-incompatible materials such as silicon or PDMS, additional steps need to be taken to render these materials more suitable for cell adhesion and maintenance. This review describes multiple surface modification strategies to improve the biocompatibility of MEMS materials. Basic concepts of cell-biomaterial interactions, such as protein adsorption and cell adhesion are covered. Finally, the applications of these MEMS materials in Tissue Engineering are presented. PMID:20054478

Ni, Ming; Tong, Wen Hao; Choudhury, Deepak; Rahim, Nur Aida Abdul; Iliescu, Ciprian; Yu, Hanry

2009-01-01

230

Cellular toxicity in Chinese hamster ovary cell cultures. I Analysis of cytotoxicity endpoints for twenty-nine priority pollutants  

Microsoft Academic Search

Chinese hamster ovary cells were exposed to 29 toxic chemical substances which were representative of several classes of compounds listed by the Natural Resources Defense Council Consent Decree as priority toxic pollutants. After cell cultures were exposed to the test substance, cell samples were assayed for protein and DNA synthesis, ATP, cell number, and viability. A filter-disk technique employing a

N. E. Garrett; J. Lewtas

1983-01-01

231

Mitigation of variation observed in a peripheral blood mononuclear cell (PBMC) based HIV-1 neutralization assay by donor cell pooling.  

PubMed

Cultured primary peripheral blood mononuclear cells (PBMC) represent a potentially physiologic in vitro model of HIV-1 infection, but assessment of antibody-mediated HIV-1 neutralization using PBMC has been hindered by donor variability and lack of a sustainable individual PBMC source. To advance this model for HIV vaccine evaluation, intra- and inter-assay variability were assessed using monoclonal and polyclonal antibodies and PBMC targets from multiple HIV-seronegative donors. Inter-assay variability was introduced by using different PBMC for virus propagation, and more substantially, for assay targets. Neutralization titers varied by as much as 4 logs when using different individual donor PBMC as targets; variability was antibody-specific, with the greatest variation observed using an individual polyclonal plasma. Pooling of multiple PBMC donors significantly reduced median inter-assay variation to the level of intra-assay variation, suggesting a pathway forward for establishing a uniform, sustainable and standardized approach to the assessment of antibody function using a PBMC model. PMID:24210120

Wieczorek, Lindsay; Brown, Bruce K; Delsarto Macedo, Camila; Wesberry-Schmierer, Maggie; Ngauy, Viseth; Rosa Borges, Andrew; Michael, Nelson L; Marovich, Mary A; Montefiori, David C; Polonis, Victoria R

2013-12-01

232

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

SciTech Connect

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.

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

1988-05-01

233

Performance of the Verigene Gram-Negative Blood Culture Assay for Rapid Detection of Bacteria and Resistance Determinants  

PubMed Central

Nonduplicate blood cultures that were positive for Gram-negative bacilli (n = 125) were tested by the Verigene Gram-negative blood culture (BC-GN) assay; 117 (90.7%) isolates were members of the panel. For identification and resistance markers, the agreements with routine methods were 97.4% (114/117) and 92.3% (12/13). The BC-GN assay is a rapid and accurate tool for the detection of pathogens from blood cultures and could be integrated alongside conventional systems to enable faster patient management, but the clinical benefits should be further evaluated. PMID:24899026

De Mendonça, Ricardo; Nonhoff, Claire; Roisin, Sandrine; Denis, Olivier

2014-01-01

234

Effect of amniotic fluid on the in vitro culture of human corneal endothelial cells.  

PubMed

The present study was designed to evaluate the effects of human amniotic fluid (HAF) on the growth of human corneal endothelial cells (HCECs) and to establish an in vitro method for expanding HCECs. HCECs were cultured in DMEM-F12 supplemented with 20% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using either FBS- or HAF-containing media. Cell proliferation and cell death ELISA assays were performed to determine the effect of HAF on cell growth and viability. The identity of the cells cultured in 20% HAF was determined using immunocytochemistry (ICC) and real-time reverse transcription polymerase chain reaction (RT-PCR) techniques to evaluate the expression of factors that are characteristic of HCECs, including Ki-67, Vimentin, Na+/K+-ATPase and ZO-1. HCEC primary cultures were successfully established using 20% HAF-containing medium, and these cultures demonstrated rapid cell proliferation according to the cell proliferation and death ELISA assay results. The ICC and real time RT-PCR results indicated that there was a higher expression of Na+/K+-ATPase and ZO-1 in the 20% HAF cell cultures compared with the control (20% FBS) (P < 0.05). The 20% HAF-containing medium exhibited a greater stimulatory effect on HCEC growth and could represent a potential enriched supplement for HCEC regeneration studies. PMID:24726921

Feizi, Sepehr; Soheili, Zahra-Soheila; Bagheri, Abouzar; Balagholi, Sahar; Mohammadian, Azam; Rezaei-Kanavi, Mozhgan; Ahmadieh, Hamid; Samiei, Shahram; Negahban, Kambiz

2014-05-01

235

Multiplex Real-Time PCR Assay for Rapid Detection of Methicillin-Resistant Staphylococci Directly from Positive Blood Cultures  

PubMed Central

Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of bloodstream infections (BSIs) and is recognized as a major nosocomial pathogen. This study aimed to evaluate a newly designed multiplex real-time PCR assay capable of the simultaneous detection of mecA, S. aureus, and coagulase-negative staphylococci (CoNS) in blood culture specimens. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays (M&D, Republic of Korea) use the TaqMan probes 16S rRNA for Staphylococcus spp., the nuc gene for S. aureus, and the mecA gene for methicillin resistance. The detection limit of the multiplex real-time PCR assay was 103 CFU/ml per PCR for each gene target. The multiplex real-time PCR assay was evaluated using 118 clinical isolates from various specimen types and a total of 350 positive blood cultures from a continuous monitoring blood culture system. The results obtained with the multiplex real-time PCR assay for the three targets were in agreement with those of conventional identification and susceptibility testing methods except for one organism. Of 350 positive bottle cultures, the sensitivities of the multiplex real-time PCR kit were 100% (166/166 cultures), 97.2% (35/36 cultures), and 99.2% (117/118 cultures) for the 16S rRNA, nuc, and mecA genes, respectively, and the specificities for all three targets were 100%. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays are very useful for the rapid accurate diagnosis of staphylococcal BSIs. In addition, the Real-MRSA and Real-MRCoNS multiplex real-time PCR assays could have an important impact on the choice of appropriate antimicrobial therapy, based on detection of the mecA gene. PMID:24648566

Wang, Hye-young; Kim, Sunghyun; Kim, Jungho; Park, Soon-Deok

2014-01-01

236

High throughput RNAi assay optimization using adherent cell cytometry  

PubMed Central

Background siRNA technology is a promising tool for gene therapy of vascular disease. Due to the multitude of reagents and cell types, RNAi experiment optimization can be time-consuming. In this study adherent cell cytometry was used to rapidly optimize siRNA transfection in human aortic vascular smooth muscle cells (AoSMC). Methods AoSMC were seeded at a density of 3000-8000 cells/well of a 96well plate. 24 hours later AoSMC were transfected with either non-targeting unlabeled siRNA (50 nM), or non-targeting labeled siRNA, siGLO Red (5 or 50 nM) using no transfection reagent, HiPerfect or Lipofectamine RNAiMax. For counting cells, Hoechst nuclei stain or Cell Tracker green were used. For data analysis an adherent cell cytometer, Celigo® was used. Data was normalized to the transfection reagent alone group and expressed as red pixel count/cell. Results After 24 hours, none of the transfection conditions led to cell loss. Red fluorescence counts were normalized to the AoSMC count. RNAiMax was more potent compared to HiPerfect or no transfection reagent at 5 nM siGLO Red (4.12 +/-1.04 vs. 0.70 +/-0.26 vs. 0.15 +/-0.13 red pixel/cell) and 50 nM siGLO Red (6.49 +/-1.81 vs. 2.52 +/-0.67 vs. 0.34 +/-0.19). Fluorescence expression results supported gene knockdown achieved by using MARCKS targeting siRNA in AoSMCs. Conclusion This study underscores that RNAi delivery depends heavily on the choice of delivery method. Adherent cell cytometry can be used as a high throughput-screening tool for the optimization of RNAi assays. This technology can accelerate in vitro cell assays and thus save costs. PMID:21518450

2011-01-01

237

Bioactive Copper-Doped Glass Scaffolds Can Stimulate Endothelial Cells in Co-Culture in Combination with Mesenchymal Stem Cells  

PubMed Central

Bioactive glass (BG) scaffolds are being investigated for bone tissue engineering applications because of their osteoconductive and angiogenic nature. However, to increase the in vivo performance of the scaffold, including enhancing the angiogenetic growth into the scaffolds, some researchers use different modifications of the scaffold including addition of inorganic ionic components to the basic BG composition. In this study, we investigated the in vitro biocompatibility and bioactivity of Cu2+-doped BG derived scaffolds in either BMSC (bone-marrow derived mesenchymal stem cells)-only culture or co-culture of BMSC and human dermal microvascular endothelial cells (HDMEC). In BMSC-only culture, cells were seeded either directly on the scaffolds (3D or direct culture) or were exposed to ionic dissolution products of the BG scaffolds, kept in permeable cell culture inserts (2D or indirect culture). Though we did not observe any direct osteoinduction of BMSCs by alkaline phosphatase (ALP) assay or by PCR, there was increased vascular endothelial growth factor (VEGF) expression, observed by PCR and ELISA assays. Additionally, the scaffolds showed no toxicity to BMSCs and there were healthy live cells found throughout the scaffold. To analyze further the reasons behind the increased VEGF expression and to exploit the benefits of the finding, we used the indirect method with HDMECs in culture plastic and Cu2+-doped BG scaffolds with or without BMSCs in cell culture inserts. There was clear observation of increased endothelial markers by both FACS analysis and acetylated LDL (acLDL) uptake assay. Only in presence of Cu2+-doped BG scaffolds with BMSCs, a high VEGF secretion was demonstrated by ELISA; and typical tubular structures were observed in culture plastics. We conclude that Cu2+-doped BG scaffolds release Cu2+, which in turn act on BMSCs to secrete VEGF. This result is of significance for the application of BG scaffolds in bone tissue engineering approaches. PMID:25470000

Rath, Subha N.; Brandl, Andreas; Hiller, Daniel; Hoppe, Alexander; Gbureck, Uwe; Horch, Raymund E.; Boccaccini, Aldo R.; Kneser, Ulrich

2014-01-01

238

A Single-Tube Real-Time PCR Assay for Mycoplasma Detection as a Routine Quality Control of Cell Therapeutics  

PubMed Central

Summary Background Contamination of cell culture and biological material by mollicute species is an important safety issue and requires testing. We have developed a singletube real-time polymerase chain reaction (PCR) assay for rapid detection of Mollicutes species stipulated by the European Pharmacopeia. Methods Primers and TaqMan probes (FAM-labeled) were deduced from 16S rDNA sequence alignment of 18 mollicutes species. A synthetic internal control (IC) DNA and an IC-specific TaqMan probe (VIC-labeled) were included. The analytical sensitivity of the assay was determined on DNA dilutions from 12 mollicute strains. Specificity was proven by the use of DNA from other bacteria. Results Analytical sensitivities of the PCR assay were in the range of 405-2,431 genomes/ml for 11 of the 12 tested mollicute DNA samples. The lowest sensitivity was found for Ureaplasma urealyticum (19,239 genomes/ml). Negative results for DNA samples from 3 different ubiquitous bacteria demonstrated the specificity of the PCR assay for Mollicutes. Direct testing of cell culture supernatants spiked with Mycoplasma orale revealed similar sensitivity compared to isolated DNA. Conclusions Our single-tube real-time PCR assay with internal reaction control enables rapid and specific detection of mollicute contaminants. The test protocol is suitable for routine quality control of cell therapeutics. PMID:24659951

Janetzko, Karin; Rink, Gabi; Hecker, Andrea; Bieback, Karen; Klüter, Harald; Bugert, Peter

2014-01-01

239

Progress Towards Drosophila Epithelial Cell Culture  

PubMed Central

Drosophila epithelial research is at the forefront of the field; however, there are no well-characterized epithelial cell lines that could provide a complementary in vitro model for studies conducted in vivo. Here, a protocol is described that produces epithelial cell lines. The method uses genetic manipulation of oncogenes or tumor suppressors to induce embryonic primary culture cells to rapidly progress to permanent cell lines. It is, however, a general method and the type of cells that comprise a given line is not controlled experimentally. Indeed, only a small fraction of the lines produced are epithelial in character. For this reason, additional work needs to be done to develop a more robust epithelial cell-specific protocol. It is expected that Drosophila epithelial cell lines will have great utility for in vitro analysis of epithelial biology, particularly high-throughput analyses such as RNAi screens. PMID:23097097

Simcox, Amanda

2015-01-01

240

The Effect of Spaceflight on Bone Cell Cultures  

NASA Technical Reports Server (NTRS)

Understanding the response of bone to mechanical loading (unloading) is extremely important in defining the means of adaptation of the body to a variety of environmental conditions such as during heightened physical activity or in extended explorations of space or the sea floor. The mechanisms of the adaptive response of bone are not well defined, but undoubtedly they involve changes occurring at the cellular level of bone structure. This proposal has intended to examine the hypothesis that the loading (unloading) response of bone is mediated by specific cells through modifications of their activity cytoskeletal elements, and/or elaboration of their extracellular matrices. For this purpose, this laboratory has utilized the results of a number of previous studies defining molecular biological, biochemical, morphological, and ultrastructural events of the reproducible mineralization of a primary bone cell (osteoblast) culture system under normal loading (1G gravity level). These data and the culture system then were examined following the use of the cultures in two NASA shuttle flights, STS-59 and STS-63. The cells collected from each of the flights were compared to respective synchronous ground (1G) control cells examined as the flight samples were simultaneously analyzed and to other control cells maintained at 1G until the time of shuttle launch, at which point they were terminated and studied (defined as basal cells). Each of the cell cultures was assayed in terms of metabolic markers- gene expression; synthesis and secretion of collagen and non-collagenous proteins, including certain cytoskeletal components; assembly of collagen into macrostructural arrays- formation of mineral; and interaction of collagen and mineral crystals during calcification of the cultures. The work has utilized a combination of biochemical techniques (radiolabeling, electrophoresis, fluorography, Western and Northern Blotting, and light microscopic immunofluorescence) and structural methods (conventional and high voltage electron microscopy, inununocytochemistry, stereomicroscopy, and 3D image reconstruction). The studies have provided new knowledge of aspects of bone cell development and structural regulation, extracellular matrix assembly, and mineralization during spaceflight and under normal gravity. The information has contributed to insights into the means in general by which cells respond and adapt to different conditions of gravity (loading). The data may as well have suggested an underlying basis for the observed loss of bone by vertebrates, including man, in microgravity; and these scientific results may have implications for understanding bone loss following fracture healing and extended periods of inactivity such as during long-term bedrest.

Landis, William J.

1999-01-01

241

Cell Cycle Progression of Human Cells Cultured in Rotating Bioreactor  

NASA Technical Reports Server (NTRS)

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.

Parks, Kelsey

2009-01-01

242

Quantitative and dynamic assay of single cell chemotaxis.  

PubMed

We have developed a single-cell assay platform that allows quantitative analysis of single cell chemotaxis by dynamic morphogenetic gradients, subcellular microscopic imaging and automated image analysis, and have applied these to measure cellular polarization of budding yeast. The computer-controlled microfluidic device regulates the gradient profile at any given time, and allows quantitative monitoring of cell morphology and the localization and expression of specific marker proteins during the dynamic polarization process. With this integrated experimental system, we compare the polarized signaling response of wild-type and far1-H7 mutant cells, which express a truncated Far1 protein unable to interact with Cdc24. Our results confirm that Far1 functions as an adaptor that recruits polarity establishment proteins to the site of extracellular signaling. Moreover, by changing the gradient profile and estimating the number of bound surface receptors, we quantitatively address why surprisingly small differences in pheromone concentration across yeast cells can be amplified into a robust polarity axis. This integrated single cell experimental platform thus opens the possibility to quantitatively investigate the molecular regulatory mechanism of chemotaxis in yeast, which serves as a paradigm to understand the fundamental processes involved in cancer metastasis, angiogenesis and axon generation. PMID:22230969

Lee, Sung Sik; Horvath, Peter; Pelet, Serge; Hegemann, Björn; Lee, Luke P; Peter, Matthias

2012-04-01

243

Effect of hawthorn (Crataegus oxycantha) crude extract and chromatographic fractions on multiple activities in a cultured cardiomyocyte assay.  

PubMed

Extracts of hawthorn (Crataegus oxycantha) have become popular herbal supplements for their well-recognized cardiotonic effects. Many commercial preparations have been used successfully in the treatment of congestive heart failure, although the active principles within these extracts have yet to be conclusively identified. Several hawthorn preparations were studied and found to have negative chronotropic effects in a cultured neonatal murine cardiomyocyte assay using unpaced cells. As compared to conventional cardiac drugs (i.e., epinephrine, milrinone, ouabain, or propranolol), hawthorn extract has a unique activity profile. Hawthorn extract appears to be anti-arrhythmic and capable of inducing rhythmicity in quiescent cardiomyocytes. Hawthorn extract does not cause beta-adrenergic receptor blockade at concentrations which cause negative chronotropic effects. Commercial hawthorn preparations, extracts prepared from dried leaves and those made from dried berries have similar chronotropic activities. When crude extracts are separated using size-exclusion chromatography, several fractions retain multiple cardiac activities. Assays with chromatographic fractions reveal that multiple dissimilar cardioactive components may exist within the extract, making the identification of individual active constituents more challenging. PMID:16487691

Long, S R; Carey, R A; Crofoot, K M; Proteau, P J; Filtz, T M

2006-11-01

244

Behaviour in culture of isolated protoplasts from “Paul's scarlet” rose suspension culture cells  

Microsoft Academic Search

Summary Protoplasts were enzymatically isolated from “Paul's scarlet” rose suspension culture cells. They were cultured in medium similar to that used to culture the cells from which they were isolated with the addition of sucrose as an osmotic stabiliser. They were studied by light and electron microscopy and their changes in size and number per culture were recorded. Expansion was

R. S. Pearce; E. C. Cocking

1973-01-01

245

Ten commandments for preventing contamination of primary cell cultures  

Microsoft Academic Search

Procedures for preventing contamination in primary cell cultures must be carefully defined and strictly followed in order to obtain healthy cells. Protocols have been developed and refined in our laboratory for establishing primary cultures of muscle and fat stem cells without contamination from a variety of animals. Contamination of cell cultures is not only frustrating, but is also very expensive

Janet L. Vierck; Katherine Byrne; Priya S. Mir; Michael V. Dodson

2000-01-01

246

Renal Tubular Cells Cultured from Genetically Modified Animals  

Microsoft Academic Search

The culture of renal tubular cells from genetically modified animals opens the opportunity of biochemical, cell biology and physiological studies under strictly controlled conditions. Either primary cultures or cell lines can be used. Through two examples of primary cultures of proximal tubular cells obtained from knock-out mice, important information about the function of proteins were obtained. Mice lacking vimentin, an

Gérard Friedlander; Isabelle Runembert; François Vrtovsnik; Fabiola Terzi

1999-01-01

247

Effect of black tea extract on herpes simplex virus-1 infection of cultured cells  

PubMed Central

Background The purpose of this investigation was to determine if black tea extract (BTE), consisting primarily of flavanol compounds called theaflavins, could inhibit herpes simplex virus type-1 (HSV-1) infection in cultured A549 (human epithelial) and Vero cells. Methods The effect of BTE both on A549 and Vero cultured cells and on HSV-1 was assessed by using phase contrast and fluorescent microscopy, and cell viability and proliferation assays. After establishing the maximum non-cytotoxic concentration of BTE, A549 and Vero cells and HSV-1 virions were treated with varying concentrations of BTE, respectively. A549 and Vero cells were infected with HSV-1 with green fluorescent protein (GFP) insert at the UL46 gene. The effect of infectivity was determined by viral DNA extraction followed by PCR, plaque assays, adsorption assays, and electrophoresis of PCR products. Results BTE was not cytotoxic to A549 and Vero cells, as confirmed by cell viability and proliferation assays, in which BTE treated groups paralleled the positive control group. For both cell lines, plaque assays and fluorescent microscopy indicated an inverse relationship between BTE concentration (from 0.14 ?M – 1.4 mM) and HSV-1 infectivity. Specifically, PCR and electrophoresis showed a reduction in the viral genome following treatment with BTE. In addition, there was a noticeable decrease in the amount of viral plaques for BTE treated samples in the adsorption assays. Conclusions BTE consisting primarily of theaflavins is not cytotoxic and can reduce or block the production of infectious HSV-1 virions in cultured A549 and Vero cells, thus inhibiting the infectivity of the virus by interfering in the attachment, penetration and viral DNA replication of HSV-1 particles. These findings indicate that BTE enriched with theaflavins has the potential to be developed as a safe, therapeutic antiviral agent to prevent the spread of HSV-1. PMID:23777309

2013-01-01

248

Neonatal rat heart cells cultured in simulated microgravity  

Microsoft Academic Search

Summary  \\u000a In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit\\u000a gravity constrain cells to propagate, differentiate, and interact in a two-dimensional (2D) plane. Neonatal rat cardiac cells\\u000a in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by nonmyocyte cell types.\\u000a Such cardiac cell cultures respond predictably

Robert E. Akins; Nancy A. Schroedl; Steve R. Gonda; Charles R. Hartzell

1997-01-01

249

Plastid transformation of tobacco suspension cell cultures.  

PubMed

Chloroplast transformation has been extremely valuable for the study of plastid biology and gene expression, but the tissue culture methodology involved can be laborious, and it can take several months to obtain homoplasmic regenerated plants useful for molecular or physiological studies. In contrast, transformation of tobacco suspension cell plastids provides an easy and efficient system to rapidly evaluate the efficacy of multiple constructs prior to plant regeneration. Suspension cell cultures can be initiated from many cell types, and once established, can be maintained by subculture for more than a year with no loss of transformation efficiency. Using antibiotic selection, homoplasmy is readily achieved in uniform cell colonies useful for comparative gene expression analyses, with the added flexibility to subsequently regenerate plants for in planta studies. Plastids from suspension cells grown in the dark are similar in size and cellular morphology to those in embryogenic culture systems of monocot species, thus providing a useful model for understanding the steps leading to plastid transformation in those recalcitrant species. PMID:24599853

Staub, Jeffrey M

2014-01-01

250

Dynamic cell culture system (7-IML-1)  

NASA Technical Reports Server (NTRS)

This experiment is one of the Biorack experiments being flown on the International Microgravity Laboratory 1 (MIL-1) mission as part of an investigation studying cell proliferation and performance in space. One of the objectives of this investigation is to assess the potential benefits of bioprocessing in space with the ultimate goal of developing a bioreactor for continuous cell cultures in space. This experiment will test the operation of an automated culture chamber that was designed for use in a Bioreactor in space. The device to be tested is called the Dynamic Cell Culture System (DCCS). It is a simple device in which media are renewed or chemicals are injected automatically, by means of osmotic pumps. This experiment uses four Type I/O experiment containers. One DCCS unit, which contains a culture chamber with renewal of medium and a second chamber without a medium supply fits in each container. Two DCCS units are maintained under zero gravity conditions during the on-orbit period. The other two units are maintained under 1 gh conditions in a 1 g centrifuge. The schedule for incubator transfer is given.

Cogoli, Augusto

1992-01-01

251

THE ACTIVITY OF ENVIRONMENTAL SAMPLES IN A CELL CULTURE TEST FOR ASBESTOS TOXICITY  

EPA Science Inventory

The inhibition of colony-forming efficiency of cultured human embryonic intestine-derived epithelial (I-407) cells was utilized in order to assay the toxic potential of six coded samples of particulate matter provided by the United States Environmental Protection Agency (EPA). Th...

252

Identification of Hedgehog Pathway Components by RNAi in Drosophila Cultured Cells  

Microsoft Academic Search

Classical genetic screens can be limited by the selectivity of mutational targeting, the complexities of anatomically based phenotypic analysis, or difficulties in subsequent gene identification. Focusing on signaling response to the secreted morphogen Hedgehog (Hh), we used RNA interference (RNAi) and a quantitative cultured cell assay to systematically screen functional roles of all kinases and phosphatases, and subsequently 43% of

Lawrence Lum; Shenqin Yao; Brian Mozer; Alessandra Rovescalli; Doris Von Kessler; Marshall Nirenberg; Philip A. Beachy

2003-01-01

253

Rosmarinic acid production in Coleus cell cultures.  

PubMed

Cell suspension cultures of Coleus blumei Benth. have been found to accumulate 8-11% of their dry weight as rosmarinic acid (?-O-caffeoyl-3,4-dihydroxyphenyl-lactic acid). Actively-growing tissue converts >20% of exogenously supplied phenylalanine and tyrosine to the caffeoyl ester and this high rate of synthesis coincides with an increase in phenylalanine ammonia-lyase specific activity. Administration to the cultures of known phenylpropanoid precursors of rosmarinic acid failed to enhance the latter's production and in some cases inhibited it. PMID:24420667

Razzaque, A; Ellis, B E

1977-01-01

254

An Immunohistochemical Method to Study Breast Cancer Cell Subpopulations and Their Growth Regulation by Hormones in Three-Dimensional Cultures  

PubMed Central

The development of in vitro three-dimensional cell culture matrices offers physiologically relevant alternatives to traditional culture on plastic surfaces. However methods to analyze cell subpopulations therein are poor. Here we present a simple and inexpensive method to analyze cell subpopulations in mixed-cell colonies using standard immunohistochemical (IHC) techniques. Briefly, Matrigel™ blocks are sandwiched between two layers of HistoGel™, hardened by rapid cooling then processed for routine fixation, paraffin embedding, and IHC. We demonstrate the assay using mono- and co-cultured normal human breast, human breast cancer, and transformed mouse stromal cells along with hormone treated breast cancer cells. Judicious selection of specific antibodies allows different cell types within heterotypic colonies to be identified. A brief pulse of bromodeoxyuridine in living colonies allows proliferation of cell subpopulations to be quantified. This simple assay is useful for multiple cell types, species, and conditions. PMID:22649363

Pinto, Mauricio P.; Jacobsen, Britta M.; Horwitz, Kathryn B.

2011-01-01

255

An immunohistochemical method to study breast cancer cell subpopulations and their growth regulation by hormones in three-dimensional cultures.  

PubMed

The development of in vitro three-dimensional cell culture matrices offers physiologically relevant alternatives to traditional culture on plastic surfaces. However methods to analyze cell subpopulations therein are poor. Here we present a simple and inexpensive method to analyze cell subpopulations in mixed-cell colonies using standard immunohistochemical (IHC) techniques. Briefly, Matrigel™ blocks are sandwiched between two layers of HistoGel™, hardened by rapid cooling then processed for routine fixation, paraffin embedding, and IHC. We demonstrate the assay using mono- and co-cultured normal human breast, human breast cancer, and transformed mouse stromal cells along with hormone treated breast cancer cells. Judicious selection of specific antibodies allows different cell types within heterotypic colonies to be identified. A brief pulse of bromodeoxyuridine in living colonies allows proliferation of cell subpopulations to be quantified. This simple assay is useful for multiple cell types, species, and conditions. PMID:22649363

Pinto, Mauricio P; Jacobsen, Britta M; Horwitz, Kathryn B

2011-01-01

256

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

SciTech Connect

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

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

257

Genomics in mammalian cell culture bioprocessing  

PubMed Central

Explicitly identifying the genome of a host organism including sequencing, mapping, and annotating its genetic code has become a priority in the field of biotechnology with aims at improving the efficiency and understanding of cell culture bioprocessing. Recombinant protein therapeutics, primarily produced in mammalian cells, constitute a $108 billion global market. The most common mammalian cell line used in biologic production processes is the Chinese hamster ovary (CHO) cell line, and although great improvements have been made in titer production over the past 25 years, the underlying molecular and physiological factors are not well understood. Confident understanding of CHO bioprocessing elements (e.g. cell line selection, protein production, and reproducibility of process performance and product specifications) would significantly improve with a well understood genome. This review describes mammalian cell culture use in bioprocessing, the importance of obtaining CHO cell line genetic sequences, and the current status of sequencing efforts. Furthermore, transcriptomic techniques and gene expression tools are presented, and case studies exploring genomic techniques and applications aimed to improve mammalian bioprocess performance are reviewed. Finally, future implications of genomic advances are surmised. PMID:22079893

Wuest, Diane M.; Harcum, Sarah W.; Lee, Kelvin H.

2013-01-01

258

An embryogenic cell suspension culture of Picea glauca (White spruce)  

Microsoft Academic Search

A cell suspension culture of Picea glauca (White spruce) which continuously produces somatic embryos has been established. Embryogenic callus derived from cultured zygotic embryos was used to initiate the culture. Numerous embryos at various early stages of development were recognized; they exhibited a meristematic embryonic region and suspensor consisting of elongate, vacuolated cells. The culture also contained clumps of meristematic

I. Hakman; L. C. Fowke

1987-01-01

259

Method of determining the number of cells in cell culture  

SciTech Connect

This patent describes a color-sensitivity method for determining the number of cells in in vitro cell culture at a sensitivity as low as about 100 or about 500 cells. It comprises lysing the cells and incubating the lysate with p-nitrophenyl phosphate at acid pH for a predetermined period of time at a temperature of from about 35{degrees} to about 38{degrees}C. and then measuring the color development at 400 to 420 nanometers and correlating the color development with cell number by comparing with a control standard of known cell number.

Connolly, D.T.

1990-06-12

260

Establishing midgut cell culture from Rhynchophorus ferrugineus (Olivier) and toxicity assessment against ten different insecticides.  

PubMed

Midgut epithelial cell culture was successfully developed from red palm weevil (Rhynchophorus ferrugineus) during this study and named as RPW-1. Optimum conditions for four different commercial media were also worked out to successfully maintain the culture. Grace's medium was found to be the most effective for RPW-1 culturing which resulted in the highest cell density of 7.5 × 10(6) cells/ml after 72 h of cell seeding with 96% cell viability. It was followed by Schneider's medium and TNM-FH medium where cell densities reached up to 7.4 × 10(6) and 5.9 × 10(6) cells/ml, respectively, after 72 h having 91 and 89% cell viability. Comparatively, Media-199 was least effective for RPW-1 cell culturing. As a whole, temperature at 27°C and pH 6.3 were the best for RPW-1 culturing where the highest cell density and maximum cell viability were noted. Individually, Grace's medium, Schneider's medium, TNM-FH medium, and Media-199 produced better results at 27°C, 27°C, 24°C, and 21°C and pH 6.3, 6.4, 5.3, and 7.1, respectively. The toxicity assay and MTT cell proliferation assay revealed that, out of the ten insecticides used in this study, emamectin benzoate was the most toxic insecticide to RPW-1 cells resulting in 92% cell mortality and 74% cell growth inhibition. Dieldrin was the least potent, causing only 19% cell mortality and 18% cell growth inhibition. PMID:24197670

Aljabr, Ahmed Mohammed; Rizwan-ul-Haq, Muhammad; Hussain, Abid; Al-Mubarak, Abdullah I; Al-Ayied, Hassan Y

2014-04-01

261

Effect of antioxidants on PDT treatment of cultured tumor cells  

NASA Astrophysics Data System (ADS)

Lipid peroxidation (LP) is involved in cell damage induced by photodynamic treatment (PDT) sensitized by some lipophylic porphyrins. We investigated an effect of lipophylic antioxidant (alpha) -tocopherol and its water-soluble analog, trolox, on meta-tetra(hydroxyphenyl)chlorin (mTHPC) sensitized PDT (413 nm) of cultured human colon adenocarcinoma cells (HT29). Cell survival was measured by the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide conversion to farmazan (MTT assay). Both antioxidants in concentrations lower than 0.1 mM did not affect photokilling of HT29 cells. These data might suggest that LP is not of crucial importance in cell damage photosensitized by mTHPC. One mM (alpha) -tocopherol or trolox decreased cell survival by ca. 15 and 13% respectively. Both antioxidants increased PDT- induced damage of HT29. Potentiation was evident as the decrease in the initial shoulder part of fluence dependence curve. We propose that antioxidants at height, pro-oxidant concentrations can potentiate PDT induced killing of tumor cells.

Melnikova, Vladislava; Bezdetnaya, Lina N.; Belitchenko, Irina; Potapenko, Alexander Y.; Merlin, Jean-Louis; Guillemin, Francois H.

1998-05-01

262

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

NASA Astrophysics Data System (ADS)

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.

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

2014-11-01

263

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

PubMed

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 enzyme replacement therapy, gene therapy, and combination therapies. PMID:23840811

Mauri, Victor; Lotfi, Parisa; Segatori, Laura; Sardiello, Marco

2013-01-01

264

Nick translation - a new assay for monitoring DNA damage and repair in cultured human fibroblasts  

SciTech Connect

An in vitro assay has been developed to detect DNA damage and repair following chemical treatment of human diploid fibroblasts. DNA damage is measured by following the Escherichia coli DNA polymerase I-catalyzed incorporation of radiolabeled deoxycytidine triphosphate (dCTP) into the DNA of lysolecithin-permeabilized cells. DNA strand breaks with free 3' OH termini serve as template sites for incorporation, and decrease of this incorporation with time, following removal of the test chemical, indicates loss (repair) of initial damage. Inhibition of the DNA excision repair process by the addition of the repair inhibitors arabinofuranosyl cytosine (ara-C) and hydroxyurea (HU) during the incubation period gives rise to an increased number of template sites, manifesting itself in increased incorporation and indicating the induction of long-patch excision repair. Results presented demonstrate that all 14 direct-acting carcinogens tested and 8 of 14 carcinogens requiring metabolic activation give positive indication of DNA damage, repair, or both. Eleven of 14 noncarcinogens tested were scored as negative, the other 3 having previously been shown to interact with cellular DNA. This assay is shown to have predictive capability at least equal to that of UDS assays but to allow a broader spectrum of genotoxic effects to be analyzed.

Snyder, R.D.; Matheson, D.W.

1985-01-01

265

UVA-induced oxidative stress in single cells probed by autofluorescence modifications, cloning assay, and comet assay  

NASA Astrophysics Data System (ADS)

Cell damage by low-power 365 nm radiation of a 50 W high-pressure mercury microscopy lamp was studied. UVA exposure to CHO cells resulted for radiant exposures greater than 10 kJ/m2 in significant modifications of NADH-attributed autofluorescence and in inhibition of cell division. Single cell gel electrophoresis (comet assay) revealed UVA-induced single strand DNA breaks. According to these results, UVA excitation radiation in fluorescence microscopy may damage cells. This has to be considered in vital cell microscopy, e.g. in calcium measurements.

Koenig, Karsten; Krasieva, Tatjana; Bauer, Eckhard; Fiedler, Ulrich; Berns, Michael W.; Tromberg, Bruce J.; Greulich, Karl O.

1996-01-01

266

Real-time PCR assays for genus-specific detection and quantification of culturable and non-culturable mycobacteria and pseudomonads in metalworking fluids  

Microsoft Academic Search

Genus-specific real-time PCR assays were developed and optimized for the direct culture-independent detection and quantification of Mycobacteria and Pseudomonads in contaminated metalworking fluids (MWF) and the results were compared with conventional culturing using selective media. It included optimization of the direct DNA isolation from the fluid matrix and the amplification conditions using genus-specific primers. Mycobacterium-specific primers based on 65-kDa heat

Izhar U. H. Khan; Jagjit S. Yadav

2004-01-01

267

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

NASA Astrophysics Data System (ADS)

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.

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

268

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

NASA Astrophysics Data System (ADS)

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.

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

2014-12-01

269

Differentiation of mammalian skeletal muscle cells cultured on microcarrier beads in a rotating cell culture system  

NASA Technical Reports Server (NTRS)

The growth and repair of adult skeletal muscle are due in part to activation of muscle precursor cells, commonly known as satellite cells or myoblasts. These cells are responsive to a variety of environmental cues, including mechanical stimuli. The overall goal of the research is to examine the role of mechanical signalling mechanisms in muscle growth and plasticity through utilisation of cell culture systems where other potential signalling pathways (i.e. chemical and electrical stimuli) are controlled. To explore the effects of decreased mechanical loading on muscle differentiation, mammalian myoblasts are cultured in a bioreactor (rotating cell culture system), a model that has been utilised to simulate microgravity. C2C12 murine myoblasts are cultured on microcarrier beads in a bioreactor and followed throughout differentiation as they form a network of multinucleated myotubes. In comparison with three-dimensional control cultures that consist of myoblasts cultured on microcarrier beads in teflon bags, myoblasts cultured in the bioreactor exhibit an attenuation in differentiation. This is demonstrated by reduced immunohistochemical staining for myogenin and alpha-actinin. Western analysis shows a decrease, in bioreactor cultures compared with control cultures, in levels of the contractile proteins myosin (47% decrease, p < 0.01) and tropomyosin (63% decrease, p < 0.01). Hydrodynamic measurements indicate that the decrease in differentiation may be due, at least in part, to fluid stresses acting on the myotubes. In addition, constraints on aggregate size imposed by the action of fluid forces in the bioreactor affect differentiation. These results may have implications for muscle growth and repair during spaceflight.

Torgan, C. E.; Burge, S. S.; Collinsworth, A. M.; Truskey, G. A.; Kraus, W. E.

2000-01-01

270

Recombinant protein production and insect cell culture and process  

NASA Technical Reports Server (NTRS)

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 the cultured insect cells as host for a virus encoding the described polypeptide such as baculovirus. The insect cells can also be a host for viral production.

Spaulding, Glenn (inventor); Prewett, Tacey (inventor); Goodwin, Thomas (inventor); Francis, Karen (inventor); Andrews, Angela (inventor); Oconnor, Kim (inventor)

1993-01-01

271

Long-Term Culture of Capillary Endothelial Cells  

Microsoft Academic Search

Capillary endothelial cells from rats, calves, and humans, have been carried in long-term culture. Bovine capillary endothelial cells have been cloned and maintained by serial passage for longer than 8 months. This prolonged culture was accomplished by using tumor-conditioned medium, gelatin-coated plates, and a method of enriching cells in primary culture. Cultured bovine capillary endothelial cells produce Factor VIII antigen

Judah Folkman; Christian C. Haudenschild; Bruce R. Zetter

1979-01-01

272

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

DOEpatents

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.

An, Yuehuei H. (Charleston, SC); Mironov, Vladimir A. (Mt. Pleasant, SC); Gutowska, Anna (Richland, WA)

2000-01-01

273

Simultaneous detection and identification of common cell culture contaminant and pathogenic mollicutes strains by reverse line blot hybridization.  

PubMed

We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Acholeplasma laidlawii) and human infection (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum). We developed a nested PCR assay with "universal" primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens. PMID:15006769

Wang, Hui; Kong, Fanrong; Jelfs, Peter; James, Gregory; Gilbert, Gwendolyn L

2004-03-01

274

Sequencing technologies for animal cell culture research.  

PubMed

Over the last 10 years, 2nd and 3rd generation sequencing technologies have made the use of genomic sequencing within the animal cell culture community increasingly commonplace. Each technology's defining characteristics are unique, including the cost, time, sequence read length, daily throughput, and occurrence of sequence errors. Given each sequencing technology's intrinsic advantages and disadvantages, the optimal technology for a given experiment depends on the particular experiment's objective. This review discusses the current characteristics of six next-generation sequencing technologies, compares the differences between them, and characterizes their relevance to the animal cell culture community. These technologies are continually improving, as evidenced by the recent achievement of the field's benchmark goal: sequencing a human genome for less than $1,000. PMID:25214225

Kremkow, Benjamin G; Lee, Kelvin H

2015-01-01

275

Cell assembly patterns of embryonic mouse cerebellar cells on carbohydrate-derivatized polylysine culture substrata  

PubMed Central

Four carbohydrate derivatives of poly-D-lysine have been synthesized and assayed as substrates for the tissue culture of embryonic mouse cerebellar cells. On poly-beta-(D-glucopyranosyl)-poly-D-lysine and on poly-beta-(N-acetyl-D-glucosaminyl)-poly-D-lysine, dissociated cerebellar cells formed a monolayer. On poly-beta-(D-galactopyranosyl)- poly-D-lysine, cellular aggregates were formed and cables of processes were extended between the aggregates. On poly-beta-(L-fucosyl)-poly-D- lysine, cerebellar cells failed to attach and died within 24 h. On poly- (N-acetyl)-poly-D-lysine, cell attachment was identical to that on poly- D-lysine. At low concentrations of underivatized poly-D-lysine (0.5-2.0 microgram/ml) dissociated embryonic cerebellar cells formed cellular aggregates, whereas at higher concentrations of poly-D-lysine monolayering was extensive. PMID:7228900

1981-01-01

276

High-Content Assays for Hepatotoxicity Using Induced Pluripotent Stem Cell–Derived Cells  

PubMed Central

Abstract Development of predictive in vitro assays for early toxicity evaluation is extremely important for improving the drug development process and reducing drug attrition rates during clinical development. High-content imaging-based in vitro toxicity assays are emerging as efficient tools for safety and efficacy testing to improve drug development efficiency. In this report we have used an induced pluripotent stem cell (iPSC)–derived hepatocyte cell model having a primary tissue-like phenotype, unlimited availability, and the potential to compare cells from different individuals. We examined a number of assays and phenotypic markers and developed automated screening methods for assessing multiparameter readouts of general and mechanism-specific hepatotoxicity. Endpoints assessed were cell viability, nuclear shape, average and integrated cell area, mitochondrial membrane potential, phospholipid accumulation, cytoskeleton integrity, and apoptosis. We assayed compounds with known mechanisms of toxicity and also evaluated a diverse hepatotoxicity library of 240 compounds. We conclude that high-content automated screening assays using iPSC-derived hepatocytes are feasible, provide information about mechanisms of toxicity, and can facilitate the safety assessment of drugs and chemicals. PMID:24229356

Sirenko, Oksana; Hesley, Jayne; Rusyn, Ivan

2014-01-01

277

Evaluation of a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay (Keystone Sym)  

EPA Science Inventory

Our goal is to establish an in vitro model system to evaluate chemical effects using a single stem cell culture technique that would improve throughput and provide quantitative markers of differentiation and cell number. To this end, we have used an adherent cell differentiation ...

278

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

PubMed Central

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 ?III-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 to validation assays. PMID:25426336

Payne, Tiffany

2014-01-01

279

Direct measurement of clathrin-coated vesicle formation using a cell-free assay.  

PubMed

Factors controlling the last stages of clathrin-coated vesicle formation were investigated using an assay allowing direct measurement of the detachment of these vesicles from the plasma membrane. Plasma membranes from cultured cells surface-labelled with 125I-alpha2-macroglobulin (a ligand that preferentially associates with clathrin-coated pits) were isolated by sonication of cells attached to a poly-L-lysine-coated substratum and incubated in the presence of nucleotide(s) +/- cytosol. A significant proportion of the membrane-associated radioactivity was released into the incubation medium in sedimentable form (14x10(6)g). The nucleotide and ligand specificities of this process together with the results of a series of biochemical, morphological and gradient analyses, led to the conclusion that measurement of the released sedimentable radioactivity provides a direct estimate of the formation of clathrin-coated vesicles from clathrin-coated pits. A morphological analysis of quick-frozen replicas of these membranes indicated that only the last stages of clathrin-coated vesicle formation were studied in the assay. Taking advantage of this cell-free system, we demonstrate that membrane-associated cytosolic factors and GTP-binding proteins, noteably dynamin, play a crucial role. Moreover, although these events can occur in the absence of ATP and Ca2+, optimal conditions for the formation of clathrin-coated vesicles require the presence of ATP, GTP and cytosol. PMID:9365281

Gilbert, A; Paccaud, J P; Carpentier, J L

1997-12-01

280

The Standard Scrapie Cell Assay: Development, Utility and Prospects  

PubMed Central

Prion diseases are a family of fatal neurodegenerative diseases that involve the misfolding of a host protein, PrPC. Measuring prion infectivity is necessary for determining efficacy of a treatment or infectivity of a prion purification procedure; animal bioassays are, however, very expensive and time consuming. The Standard Scrapie Cell Assay (SSCA) provides an alternative approach. The SSCA facilitates quantitative in vitro analysis of prion strains, titres and biological properties. Given its robust nature and potential for high throughput, the SSCA has substantial utility for in vitro characterization of prions and can be deployed in a number of settings. Here we provide an overview on establishing the SSCA, its use in studies of disease dissemination and pathogenesis, potential pitfalls and a number of remaining challenges. PMID:25602372

van der Merwe, Jacques; Aiken, Judd; Westaway, David; McKenzie, Debbie

2015-01-01

281

Synthesis and antiproliferative assay of norcantharidin derivatives in cancer cells.  

PubMed

Diels-Alder reaction between furan and maleic anhydride resulted in 5,6-dehydro norcantharidin, then norcantharidin was obtained by reduction. The substituted-carboxylic acid was condensed with N-aminothiourea in presence of phosphorus oxychloride, yielding 2-amino-1,3,4-thiadiazole derivatives. Novel norcantharidin derivatives were synthesized with acylation, then intramolecular condensation using norcantharidin (or 5,6-dehydro norcantharidin) and 2-amino- 1,3,4-thiadiazole derivatives. All the target compounds were confirmed by IR, (1)HNMR, ESI-MS and were reported for the first time. Norcantharidin derivatives antiproliferative assay was tested by MTT method against A549 and PC-3 cell lines. The results showed that all the norcantharidin derivatives displayed moderate inhibitory activities. PMID:23909288

Tu, Guo Gang; Zhan, Jian Feng; Lv, Qiao Li; Wang, Jia Qi; Kuang, Bin Hai; Li, Shao Hua

2014-06-01

282

Decitabine inhibits the cell growth of cholangiocarcinoma in cultured cell lines and mouse xenografts.  

PubMed

Decitabine (DAC), an inhibitor of DNA methyltransferase, demonstrates antitumor activities in various types of cancer. However, its therapeutic potential for cholangiocarcinoma (CCA), one of the most aggressive gastrointestinal malignancies, remains to be explored. The present study investigated the antiproliferative effects of DAC on CCA cells in vitro and in vivo. Human CCA cell lines, TFK-1 and QBC939, were used as models to investigate DAC on the cell growth and proliferation of CCA. Cell proliferation was evaluated by Cell Counting Kit-8 assay combined with clonogenic survival assay. Flow cytometry, Hoechst 33342/propidium iodide staining and green fluorescent protein-tagged MAP-LC3 detection were applied to determine cell cycle progression, apoptosis and autophagy. Nude mice with TFK-1 xenografts were evaluated for tumor growth following DAC treatment. DAC was observed to significantly suppress the proliferation of cultured TFK-1 and QBC939 cells, accompanied with enhanced apoptosis, autophagy and cell cycle arrest at G2/M phase. In TFK-1 mouse xenografts, DAC retarded the tumor growth and increased the survival of CCA tumor-bearing mice. PMID:25295073

Wang, Bing; Li, Hongbo; Yang, Rui; Zhou, Shunchang; Zou, Shengquan

2014-11-01

283

Decitabine inhibits the cell growth of cholangiocarcinoma in cultured cell lines and mouse xenografts  

PubMed Central

Decitabine (DAC), an inhibitor of DNA methyltransferase, demonstrates antitumor activities in various types of cancer. However, its therapeutic potential for cholangiocarcinoma (CCA), one of the most aggressive gastrointestinal malignancies, remains to be explored. The present study investigated the antiproliferative effects of DAC on CCA cells in vitro and in vivo. Human CCA cell lines, TFK-1 and QBC939, were used as models to investigate DAC on the cell growth and proliferation of CCA. Cell proliferation was evaluated by Cell Counting Kit-8 assay combined with clonogenic survival assay. Flow cytometry, Hoechst 33342/propidium iodide staining and green fluorescent protein-tagged MAP-LC3 detection were applied to determine cell cycle progression, apoptosis and autophagy. Nude mice with TFK-1 xenografts were evaluated for tumor growth following DAC treatment. DAC was observed to significantly suppress the proliferation of cultured TFK-1 and QBC939 cells, accompanied with enhanced apoptosis, autophagy and cell cycle arrest at G2/M phase. In TFK-1 mouse xenografts, DAC retarded the tumor growth and increased the survival of CCA tumor-bearing mice. PMID:25295073

WANG, BING; LI, HONGBO; YANG, RUI; ZHOU, SHUNCHANG; ZOU, SHENGQUAN

2014-01-01

284

T-cell costimulatory capacity of oral and skin epithelial cells in vitro: presence of suppressive activity in supernatants from skin epithelial cell cultures.  

PubMed

Oral Langerhans cells (LC) have better T-cell costimulatory capacity than skin LC. In this study factors affecting this capacity have been assessed in a mixed epithelial cell lymphocyte reaction (MELR) assay. Flow cytometry analysis of freshly recovered cells revealed major histocompatibility complex (MHC) class II molecule expression on 7.5% of the oral epithelial cells and 9.7% of the skin epithelial cells. Monoclonal anti class II antibodies significantly reduced the T-cell proliferation in the MELR. Pretreatment of skin epithelial cells with interleukin-1beta, tumour necrosis factor-alpha or interferon (IFN)-gamma did not affect the MELR proliferation, but incubation with IFNgamma significantly suppressed the T-cell response. Transfer of supernatants from cultures of skin epithelial cells and allogeneic T cells to cultures of oral epithelial cells and T cells resulted in a reduced T-cell proliferation while supernatants from oral epithelial cells and T cells did not reduce proliferation. The higher proliferation in cultures of T cells and oral epithelial cells than in cultures containing skin epithelial cells may be due to the presence of a suppressive factor in the skin epithelial cell suspensions. PMID:14871193

Hasséus, B; Jontell, M; Bergenholtz, G; Dahlgren, U I

2004-02-01

285

Recycling cultured cells for immunofluorescent labeling.  

PubMed

A method to use sequential rounds of immunofluorescent labeling in cell cultures is presented. The method is based on the utilization of a non-liquid reducing agent, sodium dithionite, in conjunction with ionic or non-ionic detergents (SDS or TX100, respectively) at room temperature. This method preserves cell morphology and substrate antigenicity, and operates through the complete extraction of most primary and secondary antibodies. Using this protocol, the sequential immunolocalization of different proteins is possible, without signal interference with previous immunolabeling rounds. In addition, the method is also useful to recycle blotted membranes in immunoblots. PMID:11479721

Espada, J; Juarranz, A; Villanueva, A; Cañete, M; Andrés, I; Stockert, J C

2001-07-01

286

Patterned polymer surfaces for cell culture applications.  

PubMed

We studied the physico/chemical effects of deep UV irradiation of polystyrene, PMMA and polycarbonate with respect to cell adhesion and protein immobilization. Photochemical modifications of the polymer surfaces yielded unstable peroxides and carboxylic acid groups. Patterned enzyme and antibody adsorbates were realized by coupling via carbodiimid activation of the COOH-moities. Hepatoma cells (HepG2) and fibroblasts (L929) adhered in the presence of serum proteins in the culture medium on the irradiated regions of the substrate without any further treatment. PMID:12451876

Welle, A; Gottwald, E; Weibezahn, K F

2002-01-01

287

Image classifiers for the cell transformation assay: a progress report  

NASA Astrophysics Data System (ADS)

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.

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

2010-02-01

288

A rapid membrane based immunobinding assay for the detection of dengue virus in tissue culture.  

PubMed

A rapid, simple dot immunoassay (DOTIA) was developed and evaluated for the detection of dengue-1 viral antigen in infected Aedes albopictus C6/36 cells. Dengue virus infected cells were solubilize in sodium dodecyl sulfate (SDS) and the lysate was pressure filtered through a hydrophobic polyvinylidene difluoride (PVDF) membrane. Viral antigen retained in the membrane was detected by a dengue-1 type specific monoclonal antibody and a peroxidase-labeled second antibody. Addition of tetramethylbenzidene (TMB) substrate produced a blue-colored precipitate which allowed for quantitation of viral antigen using a portable white light reflectance densitometer. Estimate of viral infectivity in the cell lysates tested by the DOTIA was determined by standard plaque assays and the results indicated an excellent correlation between these two methods. The dot immunoassay detected dengue viral antigen in infected C6/36 cells between days three and eight post-inoculation, depending on the titer of the inoculum. An infectivity titer of at least 10(3) plaque forming units (PFU) per ml was required to detect antigen by the DOTIA. The DOTIA also detected viral antigen in cells inoculated with twelve acute sera from known dengue-1 virus infected patients, thus demonstrating that this technique is useful for the detection and identification of dengue-1 virus from clinical specimens. PMID:7939951

Simmons, M; Jennings, G; Oprandy, J J

1993-12-01

289

Effect of triazole pesticide formulation on bovine culture cells.  

PubMed

To date, most data about the possible genotoxic effect of triazole pesticides are focused on laboratory animals resulting in limited information on further non-target organisms such as cattle. The objective of the present study was to investigate the effect of triazole (tebuconazole/prothioconazole) fungicide formulation on the induction of chromosomal aberrations (CAs), sister chromatid exchanges (SCEs) and DNA fragmentation in bovine cultured lymphocytes. Our results showed that the fungicide formulation did not induce significant number of CAs in bovine cells after 24 h treatment. Nevertheless, the dose-dependent reduction of mitotic division was observed, with the strongest effect at 30.0 ?g mL(-1) in both donors (P < 0.01 and P < 0.001, respectively). Prolonged 48 h exposure caused the increased level of breaks in treated cultures (3.0-15.0 ?g mL(-1); P < 0.05) and significant decrease in mitotic index (MI). The tested fungicide failed to produce any statistical changes in the SCE frequency neither after 24 h nor 48 h treatment. However, the significant decline of the proliferation index (PI) was observed after 24 h indicating the fungicide influence on cell cycle kinetics. Prolonged 48 h exposure caused cytotoxicity reflecting in lower PI value relative to control mainly at the highest fungicide concentrations (30.0 ?g mL(-1), P < 0.001). Using painting probes for bovine chromosomes 1, 5 and 7 (BTA1, BTA5 and BTA7) only low levels of aneuploidies were detected. Significant increase of polyploidy cells (P < 0.05) was induced by a 3.0 ?g mL(-1) dose of the fungicide after 48 h. DNA fragmentation assay didn't reveal the presence of DNA nucleosome ladder in cell cultures at any time (24 h and 48 h) and fungicide concentration. PMID:24007485

Hole?ková, Beáta; Šiviková, Katarína; Dianovský, Ján; Galdíková, Martina

2013-01-01

290

A Real-Time PCR Assay for the Detection of Campylobacter jejuni in Foods after Enrichment Culture  

PubMed Central

A real-time PCR assay was developed for the quantitative detection of Campylobacter jejuni in foods after enrichment culture. The specificity of the assay for C. jejuni was demonstrated with a diverse range of Campylobacter species, related organisms, and unrelated genera. The assay had a linear range of quantification over six orders of magnitude, and the limit of detection was approximately 12 genome equivalents. The assay was used to detect C. jejuni in both naturally and artificially contaminated food samples. Ninety-seven foods, including raw poultry meat, offal, raw shellfish, and milk samples, were enriched in blood-free Campylobacter enrichment broth at 37°C for 24 h, followed by 42°C for 24 h. Enrichment cultures were subcultured to Campylobacter charcoal-cefoperazone-deoxycholate blood-free selective agar, and presumptive Campylobacter isolates were identified with phenotypic methods. DNA was extracted from enrichment cultures with a rapid lysis method and used as the template in the real-time PCR assay. A total of 66 samples were positive for C. jejuni by either method, with 57 samples positive for C. jejuni by subculture to selective agar medium and 63 samples positive in the real-time PCR assay. The results of both methods were concordant for 84 of the samples. The total time taken for detection from enrichment broth samples was approximately 3 h for the real-time PCR assay, with the results being available immediately at the end of PCR cycling, compared to 48 h for subculture to selective agar. This assay significantly reduces the total time taken for the detection of C. jejuni in foods and is an important model for other food-borne pathogens. PMID:12620820

Sails, Andrew D.; Fox, Andrew J.; Bolton, Frederick J.; Wareing, David R. A.; Greenway, David L. A.

2003-01-01

291

Highly reproducible quantification of apoptotic cells using micropatterned culture of neurons.  

PubMed

The quantification of apoptotic cells is an integral component of many cell-based assays in biological studies. However, current methods for quantifying apoptotic cells using conventional random cultures have shown great limitations, especially for the quantification of primary neurons. Randomly distributed neurons under primary culture conditions can lead to biased estimates, and vastly different estimates of cell numbers can be produced within the same experiment. In this study, we developed a simple, accurate, and reliable technique for quantifying apoptotic neurons by means of micropatterned cell cultures. A polydimethylsiloxane (PDMS) microstencil was used as a physical mask for micropatterning cell cultures, and primary granular neurons (GNs) were successfully cultured within the micropattern-confined regions and homogeneously distributed over the entire field of each pattern. As compared with the conventional method based on random cultures, the micropatterned culture method allowed for highly reproducible quantification of apoptotic cells. These results were also confirmed by using GNs derived from mice with neurodegeneration. We hope that this micropatterning method based on the use of a PDMS microstencil can overcome the technical obstacles existing in current biological studies and will serve as a powerful tool for facilitating the study of apoptosis-involved diseases. PMID:25277814

Lee, Hyun; Kim, Gyu Man; Choi, Jin Ho; Lee, Jong Kil; Bae, Jae-Sung; Jin, Hee Kyung

2015-01-15

292

Performance of enzymatic fuel cell in cell culture.  

PubMed

Here we present the very first study of an enzymatic fuel cell (EFC) in a cell culture. An EFC with Corynascus thermophilus cellobiose dehydrogenase (CDH) based bioanode and Myrothecium verrucaria bilirubin oxidase (BOx) based biocathode was constructed at the bottom of a medusa cell culture plate. The constructed EFC had a power density of up to 25 ?W cm(-2) at 0.5 V potential in simple buffer solution and in cell culturing medium. L929 murine fibroblast cells were seeded on top of the EFC and possible effects of the EFC on the cells and vice versa were studied. It was shown that on average the power of the EFC drops by about 70% under a nearly confluent layer of cells. The EFC appeared to have a toxic effect on the L929 cell line. It was concluded that the bioanode, consisting of CDH, produced hydrogen peroxide at toxic concentrations. However, the toxic effect was circumvented by co-immobilizing catalase on the bioanode. PMID:24374299

Lamberg, P; Shleev, S; Ludwig, R; Arnebrant, T; Ruzgas, T

2014-05-15

293

Cytopathogenicity of Naegleria for cultured neuroblastoma cells  

SciTech Connect

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.

Fulford, D.E.

1985-01-01

294

Comparison between clonogenic and cytotoxic assays for measuring LAK cell activity.  

PubMed

The antiproliferative effect of lymphokine-activated killer (LAK) cells was studied using a clonogenic assay in an attempt to find a model for predicting this effect in vivo or ex vivo (in the case of purging) in cancer treatment. The results were compared with the standard 51Cr-release cytotoxic assay. Cells from clonogenic neoplastic cell lines (K562 and HL-60) were plated in methylcellulose with LAK cells obtained from ten different donors in various effector-to-target (E:T) ratios. At E:T ratios of 16:1, elimination of greater than 90% of the clonogenic cells was seen in 20 of 21 experiments, whereas such lysis was incidentally found in the 51Cr-release assay. In almost all paired combinations, clonogenic cells tested in a colony assay were more sensitive to kill by LAK cells than the whole tumor cell suspensions measured in the 51Cr-release assay. PMID:2768841

Kluin-Nelemans, H C; van der Harst, D; van den Burgh, J F; van Luxemburg, S; Brand, A

1989-07-01

295

Probiotic modulation of dendritic cells co-cultured with intestinal epithelial cells  

PubMed Central

AIM: To investigate cytokine production and cell surface phenotypes of dendritic cells (DC) in the presence of epithelial cells stimulated by probiotics. METHODS: Mouse DC were cultured alone or together with mouse epithelial cell monolayers in normal or inverted systems and were stimulated with heat-killed probiotic bacteria, Bifidobacterium lactis AD011 (BL), Bifidobacterium bifidum BGN4 (BB), Lactobacillus casei IBS041 (LC), and Lactobacillus acidophilus AD031 (LA), for 12 h. Cytokine levels in the culture supernatants were determined by enzyme-linked immunosorbent assay and phenotypic analysis of DC was investigated by flow cytometry. RESULTS: BB and LC in single-cultured DC increased the expression of I-Ad, CD86 and CD40 (I-Ad, 18.51 vs 30.88, 46.11; CD86, 62.74 vs 92.7, 104.12; CD40, 0.67 vs 6.39, 3.37, P < 0.05). All of the experimental probiotics increased the production of inflammatory cytokines, interleukin (IL)-6 and tumor necrosis factor (TNF)-?. However, in the normal co-culture systems, LC and LA decreased the expression of I-Ad (39.46 vs 30.32, 33.26, P < 0.05), and none of the experimental probiotics increased the levels of IL-6 or TNF-?. In the inverted co-culture systems, LC decreased the expression of CD40 (1.36 vs -2.27, P < 0.05), and all of the experimental probiotics decreased the levels of IL-6. In addition, BL increased the production of IL-10 (103.8 vs 166.0, P < 0.05) and LC and LA increased transforming growth factor-? secretion (235.9 vs 618.9, 607.6, P < 0.05). CONCLUSION: These results suggest that specific probiotic strains exert differential immune modulation mediated by the interaction of dendritic cells and epithelial cells in the homeostasis of gastrointestinal tract. PMID:22493544

Kim, Ji Yeun; Park, Myeong Soo; Ji, Geun Eog

2012-01-01

296

Clinical Impact of a PCR Assay for Identification of Staphylococcus aureus and Determination of Methicillin Resistance Directly from Blood Cultures  

Microsoft Academic Search

We evaluated the clinical usefulness of a PCR assay that discriminates Staphylococcus aureus from coagulase- negative staphylococci and detects methicillin resistance on blood cultures by measuring the adaptation of antimicrobial therapy based on the PCR results. Only 7 of 28 patients (25%) benefited from a modification of antibiotic therapy based on the PCR results, since empirical therapy was appropriate in

M. Hallin; N. Maes; B. Byl; F. Jacobs; Y. De Gheldre; M. J. Struelens

2003-01-01

297

Cell-Based Protein Stabilization Assays for the Detection of Interactions between Small-Molecule Inhibitors and BRD4.  

PubMed

Bromodomain protein 4 (BRD4), a member of the bromodomain and extra-terminal (BET) protein family, acts as a central element in transcriptional elongation and plays essential roles in cell proliferation. Inhibition of BRD4 binding to acetylated histone tails via its two bromodomains, BD1 and BD2, with small-molecule inhibitors has been shown to be a valid strategy to prevent cancer growth. We have evaluated and established two novel assays that quantify the interaction of transfected BRD4 BD1 with chemical inhibitors inside cultured cells. Both methods are based on the principle of ligand-induced protein stabilization by which the binding of a small-molecule inhibitor stabilizes intracellular BRD4 BD1 and protects it from proteolytic degradation. We demonstrate the universal character of this principle by using two orthogonal, highly sensitive detection technologies for the quantification of BRD4 BD1 levels in cellular lysates: enzyme fragment complementation and time-resolved fluorescence resonance energy transfer (TR-FRET). Upon optimization of both assays to a miniaturized high-throughput format, the methods were validated by testing a set of small-molecule BET inhibitors and comparing the results with those from a cell-free binding assay and a biophysical thermal shift assay. In addition, point mutations were introduced into BRD4 BD1, and the corresponding mutants were characterized in the TR-FRET stabilization assay. PMID:25266565

Schulze, Jessica; Moosmayer, Dieter; Weiske, Joerg; Fernández-Montalván, Amaury; Herbst, Christopher; Jung, Marie; Haendler, Bernard; Bader, Benjamin

2015-02-01

298

Isolation, culture, and characterization of human pancreatic duct cells.  

PubMed

To establish a suitable control for pancreatic tumor cell lines, we have isolated and cultured primary human pancreatic duct cells from transplant donors. Duct cells were isolated by dissecting the main pancreatic duct and first-degree branches and enzymatic digestion. Aggregates of cells were cultured for 1 up to 5 weeks and monitored for changes in morphology and growth by phase contrast microscopy. Contaminating fibroblasts were mechanically removed from day 4 on and by cloning of epithelial cells. Cultured cells were characterized by phase contrast microscopy, electron microscopy, and immunofluorescence with antibodies against intermediate filaments (cytokeratins, vimentin, desmin), mucins (Du-Pan-2, CA 19-9), carbonic anhydrase II, acinar cell enzymes (amylase, lipase, trypsin), and islet cells. About 90% of the cultured cells could be identified as ductal epithelial cells by their expression of cytokeratins, mucins, and carbonic anhydrase II. These cells showed the ultrastructural features of duct cells. After 3-5 weeks of culture, most of the cultured cells showed co-expression of cytokeratins and vimentin in addition to duct cell markers. About 10% of cells were contaminating fibroblasts (vimentin positive, cytokeratin negative). The cultured normal human duct cells as the postulated cells of origin of the pancreatic adenocarcinoma may serve as a useful control for cultured pancreatic tumor cell lines. PMID:8460098

Trautmann, B; Schlitt, H J; Hahn, E G; Löhr, M

1993-03-01

299

Alkaline phosphatase and phosphotyrosine phosphatase activities of cultured amniotic cells with trisomy 18.  

PubMed

In cultured amniotic cells from fetuses with Edward's syndrome (trisomy 18), the activities of two protein phosphatases, alkaline phosphatase and phosphotyrosine phosphatase, were measured. Comparison with normal fetal cells showed a different behavior for each enzyme. Alkaline phosphatase was significantly lowered while phosphotyrosine phosphatase remained at normal levels. The interest of these enzyme assays in the screening procedure of this severe chromosome defect is discussed. PMID:1308852

Vergnes, H; Grozdea, J; Brisson-Lougarre, A; Bourrouillou, G; Colombies, P

1992-01-01

300

Pneumocystis jirovecii Can Be Productively Cultured in Differentiated CuFi-8 Airway Cells  

PubMed Central

ABSTRACT Although Pneumocystis jirovecii is a well-known and serious pathogen, all previous attempts to isolate, cultivate, and propagate this fungus have failed. This serious challenge in microbiology was addressed in the present study. We examined whether P. jirovecii could be cultured in a permanent three-dimensional air-liquid interface culture system formed by CuFi-8 cells, a differentiated pseudostratified airway epithelial cell line. Cultured pseudostratified cells were inoculated with bronchoalveolar fluid that had been confirmed to be positive for P. jirovecii using PCR. Five days later, the cells and basal medium were harvested and tested for P. jirovecii using quantitative PCR (qPCR), commercially available immunofluorescence detection assays, and Grocott staining of formalin-fixed, paraffin-embedded thin sections of infected-cell cultures. We successfully productively cultivated and propagated P. jirovecii from these P. jirovecii-positive bronchoalveolar lavage fluid (BALF) samples. Furthermore, we provide evidence that P. jirovecii induced cytopathic effects on lung epithelial cells and was even invasive in cell culture. To the best of our knowledge, the cell culture system developed herein represents the first methodology to enable molecular analyses of this pathogen’s life cycle and further in vitro studies of P. jirovecii, such as assessments of drug sensitivity and resistance as well as investigations of the pathogen’s stability against environmental factors and disinfectants. PMID:24825015

Schildgen, Verena; Mai, Stephanie; Khalfaoui, Soumaya; Lüsebrink, Jessica; Pieper, Monika; Tillmann, Ramona L.; Brockmann, Michael

2014-01-01

301

Enrichment of prostate cancer stem cells from primary prostate cancer cultures of biopsy samples  

PubMed Central

This study was to enrich prostate cancer stem cells (PrCSC) from primary prostate cancer cultures (PPrCC). Primary prostate cancer cells were amplified in keratinocyte serum-free medium with epidermal growth factor (EGF) and bovine pituitary extract (BPE), supplemented with leukemia inhibitory factor (LIF), stem cell factor (SCF) and cholera toxin. After amplification, cells were transferred into ultra-low attachment dishes with serum-free DMEM/F12 medium, supplemented with EGF, basic fibroblast growth factor (bFGF), bovine serum albumin (BSA), insulin, and N2 nutrition. Expression of cell-type-specific markers was determined by RT-qPCR and immunostaining. Tumorigenicity of enriched PrCSC was determined by soft agar assay and xenograft assay in NOD/SCID mice. Biopsy samples from 19 confirmed prostate cancer patients were used for establishing PPrCC, and 18 cases (95%) succeeded. Both basal marker (CK5) and luminal markers (androgen receptor and CK8) strongly co-expressed in most of PPrCC, indicating their basal epithelial origin. After amplification under adherent culture condition in vitro, transient amplifying cells were the dominant cells. Sphere formation efficiency (SFE) of passaged PPrCC was about 0.5%, which was 27 times lower than SFE of LNCaP (13.67%) in the same condition. Compared with adherent cells from PPrCC, prostasphere from PPrCC showed up regulated stem cell markers and increased tumorigenic potential in soft-agar assay. However, spheroid cells from PPrCC prostasphere failed to initiate tumor in xenograft assay in 6 months. Thus, PPrCC can be established and amplified from prostate cancer biopsy samples. Our modified sphere culture system can enrich PrCSC from PPrCC. PMID:24427338

Wang, Shunqi; Huang, Shengsong; Zhao, Xin; Zhang, Qimin; Wu, Min; Sun, Feng; Han, Gang; Wu, Denglong

2014-01-01

302

Enrichment of prostate cancer stem cells from primary prostate cancer cultures of biopsy samples.  

PubMed

This study was to enrich prostate cancer stem cells (PrCSC) from primary prostate cancer cultures (PPrCC). Primary prostate cancer cells were amplified in keratinocyte serum-free medium with epidermal growth factor (EGF) and bovine pituitary extract (BPE), supplemented with leukemia inhibitory factor (LIF), stem cell factor (SCF) and cholera toxin. After amplification, cells were transferred into ultra-low attachment dishes with serum-free DMEM/F12 medium, supplemented with EGF, basic fibroblast growth factor (bFGF), bovine serum albumin (BSA), insulin, and N2 nutrition. Expression of cell-type-specific markers was determined by RT-qPCR and immunostaining. Tumorigenicity of enriched PrCSC was determined by soft agar assay and xenograft assay in NOD/SCID mice. Biopsy samples from 19 confirmed prostate cancer patients were used for establishing PPrCC, and 18 cases (95%) succeeded. Both basal marker (CK5) and luminal markers (androgen receptor and CK8) strongly co-expressed in most of PPrCC, indicating their basal epithelial origin. After amplification under adherent culture condition in vitro, transient amplifying cells were the dominant cells. Sphere formation efficiency (SFE) of passaged PPrCC was about 0.5%, which was 27 times lower than SFE of LNCaP (13.67%) in the same condition. Compared with adherent cells from PPrCC, prostasphere from PPrCC showed up regulated stem cell markers and increased tumorigenic potential in soft-agar assay. However, spheroid cells from PPrCC prostasphere failed to initiate tumor in xenograft assay in 6 months. Thus, PPrCC can be established and amplified from prostate cancer biopsy samples. Our modified sphere culture system can enrich PrCSC from PPrCC. PMID:24427338

Wang, Shunqi; Huang, Shengsong; Zhao, Xin; Zhang, Qimin; Wu, Min; Sun, Feng; Han, Gang; Wu, Denglong

2014-01-01

303

Human preleukaemia cell culture studies in sideroblastic anaemia  

Microsoft Academic Search

Cell structure abnormalties are found in acute leukaemia and preleukaemic states. Studies on bone marrow cells and peripheral leucocytes of 4 patients with idiopathic acquired sideroblastic anaemia showed patterns in cell culture similar to those reported in acute leukaemia: 2 of these patients later developed leukaemia. Other patients with idiopathic, secondary or congenital sideroblastosis showed no such cell culture abnormalities,

J S Senn; P H Pinkerton; G B Price; T W Mak; E A McCulloch

1976-01-01

304

Cardiac Cells Beating in Culture: A Laboratory Exercise  

ERIC Educational Resources Information Center

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…

Weaver, Debora

2007-01-01

305

Cell co-culture patterning using aqueous two-phase systems.  

PubMed

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type. PMID:23567187

Frampton, John P; White, Joshua B; Abraham, Abin T; Takayama, Shuichi

2013-01-01

306

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

NASA Astrophysics Data System (ADS)

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.

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

2011-07-01

307

Equipment for large-scale mammalian cell culture.  

PubMed

This chapter provides information on commonly used equipment in industrial mammalian cell culture, with an emphasis on bioreactors. The actual equipment used in the cell culture process can vary from one company to another, but the main steps remain the same. The process involves expansion of cells in seed train and inoculation train processes followed by cultivation of cells in a production bioreactor. Process and equipment options for each stage of the cell culture process are introduced and examples are provided. Finally, the use of disposables during seed train and cell culture production is discussed. PMID:24429549

Ozturk, Sadettin S

2014-01-01

308

Microfluidic Probe for Single-Cell Lysis and Analysis in Adherent Tissue Culture  

PubMed Central

Single-cell analysis provides information critical to understanding key disease processes that are characterized by significant cellular heterogeneity. Few current methods allow single-cell measurement without removing cells from the context of interest, which not only destroys contextual information but also may perturb the process under study. Here we present a microfluidic probe that lyses single adherent cells from standard tissue culture and captures the contents to perform single-cell biochemical assays. We use this probe to measure kinase and housekeeping protein activities, separately or simultaneously, from single human hepatocellular carcinoma cells in adherent culture. This tool has the valuable ability to perform measurements that clarify connections between extracellular context, signals and responses, especially in cases where only a few cells exhibit a characteristic of interest. PMID:24594667

Lauffenburger, Douglas A.; Han, Jongyoon

2014-01-01

309

Neonatal rat heart cells cultured in simulated microgravity  

NASA Technical Reports Server (NTRS)

In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two dimensional (2D) plane. Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by non-myocyte cell types. Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system. The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue. We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity. The NASA designed High-Aspect-Ratio-Vessel (HARV) bioreactors provide a low shear environment which allows cells to be cultured in static suspension. HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques. Both systems were uniformly inoculated and medium exchanged at standard intervals. Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization. Cells in cultured in HARV's adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type. In addition, cultures prepared in HARV's using alternative support matrices also displayed morphological formations not seen in control cultures. Generally, the cells prepared in HARV and control cultures were similar, however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissue-like organizations of cardiac cells in simulated microgravity.

Akins, Robert E.; Schroedl, Nancy A.; Gonda, Steve R.; Hartzell, Charles R.

1994-01-01

310

Axitinib affects cell viability and migration of a primary foetal lung adenocarcinoma culture.  

PubMed

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-1? 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

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

311

An efficient method to isolate and culture mouse Kupffer cells.  

PubMed

Kupffer cells (KCs) play an essential role in the physiological and pathological functions of the liver. Although the isolation methods of KCs have been well-described, most of them are sophisticated and time-consuming. In addition, these methods are mainly used for isolating the KCs of the human and rat. In this study, a three-step procedure was applied to isolate KCs in sufficient number and purity from mouse liver, including the techniques of enzymatic tissue treatment, gradient centrifugation, and selective adherence. F4/80 immunofluorescence and flow cytometry were used for cell identification. The combination method resulted in a satisfactorily high yield of 5-6×10(6) KCs per liver, over 92.0% positive for F4/80 and 98.5% viable cells. After 24h of culturing, the KCs showed typical macrophage morphologic features such as irregular shape, transparent cytoplasm and kidney-like nucleus. The phagocytic assay showed that the isolated cells exhibited strong phagocytosis activity. The KCs we isolated were functionally intact and exhibited a concentration dependent TNF-? production induced by LPS. The method we described is an effective method to isolate mouse KCs in high purity and yield, which consuming fewer collagenase and time without altering the functional capacity of the KCs. PMID:24333337

Li, Pei-zhi; Li, Jin-zheng; Li, Min; Gong, Jian-ping; He, Kun

2014-01-01

312

Detection of DNA damages and repair in human culture cells with simulated space radiation  

NASA Astrophysics Data System (ADS)

DNA damages and its repair of cultured WI38 human fibroblast cells and T98G human glioblastoma cells were studied by exposing to carbon ion beams of HIMAC accelerator. The exposed cells were incubated at 37 °C for appropriate intervals and the damages were analyzed by alkaline comet assay and quantitative RT-PCR with p53 mRNA Highly inhomogeneous DNA damages were observed among the electrophoretic cell images of the comet assay. The degree of the damages was analyzed semi-quantitatively by using the Comet Index. The damaged fraction of WI38 cells was 85% immediately after 4 Gy (100 keV/?m) irradiation and decreased to 50% after 120 min. incubation indicating a repair of cell DNA. Time dependent p53 gene expression was also analyzed by the quantitative RT-PCR method.

Nagaoka, S.; Nakano, T.; Endo, S.; Onizuka, T.; Kagawa, Y.; Fujitaka, K.; Ohnishi, K.; Takahashi, A.; Ohnishi, T.

1999-09-01

313

The effects of poly L-lactic acid nanofiber scaffold on mouse spermatogonial stem cell culture  

PubMed Central

Introduction A 3D-nanofiber scaffold acts in a similar way to the extracellular matrix (ECM)/basement membrane that enhances the proliferation and self-renewal of stem cells. The goal of the present study was to investigate the effects of a poly L-lactic acid (PLLA) nanofiber scaffold on frozen-thawed neonate mouse spermatogonial stem cells (SSCs) and testis tissues. Methods The isolated spermatogonial cells were divided into six culture groups: (1) fresh spermatogonial cells, (2) fresh spermatogonial cells seeded onto PLLA, (3) frozen-thawed spermatogonial cells, (4) frozen-thawed spermatogonial cells seeded onto PLLA, (5) spermatogonial cells obtained from frozen-thawed testis tissue, and (6) spermatogonial cells obtained from frozen-thawed testis tissue seeded onto PLLA. Spermatogonial cells and testis fragments were cryopreserved and cultured for 3 weeks. Cluster assay was performed during the culture. The presence of spermatogonial cells in the culture was determined by a reverse transcriptase polymerase chain reaction for spermatogonial markers (Oct4, GFR?-1, PLZF, Mvh(VASA), Itg?6, and Itg?1), as well as the ultrastructural study of cell clusters and SSCs transplantation to a recipient azoospermic mouse. The significance of the data was analyzed using the repeated measures and analysis of variance. Results The findings indicated that the spermatogonial cells seeded on PLLA significantly increased in vitro spermatogonial cell cluster formations in comparison with the control groups (culture of SSCs not seeded on PLLA) (P?0.001). The viability rate for the frozen cells after thawing was 63.00% ± 3.56%. This number decreased significantly (40.00% ± 0.82%) in spermatogonial cells obtained from the frozen-thawed testis tissue. Both groups, however, showed in vitro cluster formation. Although the expression of spermatogonial markers was maintained after 3 weeks of culture, there was a significant downregulation for some spermatogonial genes in the experimental groups compared with those of the control groups. Furthermore, transplantation assay and transmission electron microscopy studies suggested the presence of SSCs among the cultured cells. Conclusion Although PLLA can increase the in vitro cluster formation of neonate fresh and frozen-thawed spermatogonial cells, it may also cause them to differentiate during cultivation. The study therefore has implications for SSCs proliferation and germ cell differentiation in vitro. PMID:24348035

Eslahi, Neda; Hadjighassem, Mahmoud Reza; Joghataei, Mohammad Taghi; Mirzapour, Tooba; Bakhtiyari, Mehrdad; Shakeri, Malak; Pirhajati, Vahid; Shirinbayan, Peymaneh; Koruji, Morteza

2013-01-01

314

Enumeration of water-borne bacteria using viability assays and flow cytometry: a comparison to culture-based techniques.  

PubMed

Maintaining optimal conditions in catchments or distribution systems relies heavily on water authorities having access to rapid and accurate water quality data, including an indication of bacteriological quality. In this study, the BacLight bacterial viability kit and carboxyfluorescein diacetate (CFDA) were coupled with flow cytometry (FCM) for rapid detection of physiologically active bacteria from raw and potable waters taken from various locations around South Australia. Results were compared to the direct viable count (DVC) and quantitative DVC (qDVC), in addition to the culture-based methods of the heterotrophic plate count (HPC) and a commercial SimPlate technique. Raw and potable water analysis revealed that DVC and culture-based techniques reported significantly fewer viable bacteria compared to the number of physiologically active bacteria detected using the rapid FCM assays, where this difference appeared to be nonlinear across different samples. Inconclusive results were obtained using qDVC as a viability assay. In particular, HPC results were 2-4 log orders of magnitude below that reported by the FCM assays for raw waters. Few bacteria in potable waters examined were culturable by HPC, even though FCM assays reported between 5.56 x 10(2) and 3.94 x 10(4) active bacteria ml(-1). These differences may be attributed to the presence of nonheterotrophic bacteria, sublethal injury or the adoption of an active but nonculturable (ABNC) state. PMID:14607402

Hoefel, Daniel; Grooby, Warwick L; Monis, Paul T; Andrews, Stuart; Saint, Christopher P

2003-12-01

315

Retrotransposition of marked SVA elements by human L1s in cultured cells.  

PubMed

Human retrotransposons generate structural variation and genomic diversity through ongoing retrotransposition and non-allelic homologous recombination. Cell culture retrotransposition assays have provided great insight into the genomic impact of retrotransposons, in particular, LINE-1(L1) and Alu elements; however, no such assay exists for the youngest active human retrotransposon, SINE-VNTR-Alu (SVA). Here we report the development of an SVA cell culture retrotransposition assay. We marked several SVAs with either neomycin or EGFP retrotransposition indicator cassettes. Engineered SVAs retrotranspose using L1 proteins supplemented in trans in multiple cell lines, including U2OS osteosarcoma cells where SVA retrotransposition is equal to that of an engineered L1. Engineered SVAs retrotranspose at 1-54 times the frequency of a marked pseudogene in HeLa HA cells. Furthermore, our data suggest a variable requirement for L1 ORF1p for SVA retrotransposition. Recovered engineered SVA insertions display all the hallmarks of LINE-1 retrotransposition and some contain 5' and 3' transductions, which are common for genomic SVAs. Of particular interest is the fact that four out of five insertions recovered from one SVA are full-length, with the 5' end of these insertions beginning within 5 nt of the CMV promoter transcriptional start site. This assay demonstrates that SVA elements are indeed mobilized in trans by L1. Previously intractable questions regarding SVA biology can now be addressed. PMID:21636526

Hancks, Dustin C; Goodier, John L; Mandal, Prabhat K; Cheung, Ling E; Kazazian, Haig H

2011-09-01

316

Retrotransposition of marked SVA elements by human L1s in cultured cells  

PubMed Central

Human retrotransposons generate structural variation and genomic diversity through ongoing retrotransposition and non-allelic homologous recombination. Cell culture retrotransposition assays have provided great insight into the genomic impact of retrotransposons, in particular, LINE-1(L1) and Alu elements; however, no such assay exists for the youngest active human retrotransposon, SINE-VNTR-Alu (SVA). Here we report the development of an SVA cell culture retrotransposition assay. We marked several SVAs with either neomycin or EGFP retrotransposition indicator cassettes. Engineered SVAs retrotranspose using L1 proteins supplemented in trans in multiple cell lines, including U2OS osteosarcoma cells where SVA retrotransposition is equal to that of an engineered L1. Engineered SVAs retrotranspose at 1–54 times the frequency of a marked pseudogene in HeLa HA cells. Furthermore, our data suggest a variable requirement for L1 ORF1p for SVA retrotransposition. Recovered engineered SVA insertions display all the hallmarks of LINE-1 retrotransposition and some contain 5? and 3? transductions, which are common for genomic SVAs. Of particular interest is the fact that four out of five insertions recovered from one SVA are full-length, with the 5? end of these insertions beginning within 5 nt of the CMV promoter transcriptional start site. This assay demonstrates that SVA elements are indeed mobilized in trans by L1. Previously intractable questions regarding SVA biology can now be addressed. PMID:21636526

Hancks, Dustin C.; Goodier, John L.; Mandal, Prabhat K.; Cheung, Ling E.; Kazazian, Haig H.

2011-01-01

317

Biology on a Chip: Microfabrication for Studying the Behavior of Cultured Cells  

PubMed Central

The ability to culture cells in vitro has revolutionized hypothesis testing in basic cell and molecular biology research and has become a standard methodology in drug screening and toxicology assays. However, the traditional cell culture methodology—consisting essentially of the immersion of a large population of cells in a homogeneous fluid medium—has become increasingly limiting, both from a fundamental point of view (cells in vivo are surrounded by complex spatiotemporal microenvironments) and from a practical perspective (scaling up the number of fluid handling steps and cell manipulations for high-throughput studies in vitro is prohibitively expensive). Micro fabrication technologies have enabled researchers to design, with micrometer control, the biochemical composition and topology of the substrate, the medium composition, as well as the type of neighboring cells surrounding the microenvironment of the cell. In addition, microtechnology is conceptually well suited for the development of fast, low-cost in vitro systems that allow for high-throughput culturing and analysis of cells under large numbers of conditions. Here we review a variety of applications of microfabrication in cell culture studies, with an emphasis on the biology of various cell types. PMID:15139302

Li, Nianzhen; Tourovskaia, Anna; Folch, Albert

2013-01-01

318

Oxygenation of intensive cell-culture system.  

PubMed

The abilities of various methods of oxygenation to meet the demands of high-cell-density culture were investigated using a spin filter perfusion system in a bench-top bioreactor. Oxygen demand at high cell density could not be met by sparging with air inside a spin filter (oxygen transfer values in this condition were comparable with those for surface aeration). Sparging with air outside a spin filter gave adequate oxygen transfer for the support of cell concentrations above 10(7) ml-1 in fully aerobic conditions but the addition of antifoam to control foaming caused blockage of the spinfilter mesh. Bubble-free aeration through immersed silicone tubing with pure oxygen gave similar oxygen transfer rates to that of sparging with air but without the problems of bubble damage and fouling of the spin filter. A supra-optimal level of dissolved oxygen (478% air saturation) inhibited cell growth. However, cells could recover from this stress and reach high density after reduction of the dissolved oxygen level to 50% air saturation. PMID:8590652

Emery, A N; Jan, D C; al-Rubeai, M

1995-11-01

319

Cell-type-specific level of DNA nucleotide excision repair in primary human mammary and ovarian epithelial cell cultures  

PubMed Central

DNA repair, a fundamental function of cellular metabolism, has long been presumed to be constitutive and equivalent in all cells. However, we have previously shown that normal levels of nucleotide excision repair (NER) can vary by 20-fold in a tissue-specific pattern. We have now successfully established primary cultures of normal ovarian tissue from seven women by using a novel culture system originally developed for breast epithelial cells. Epithelial cells in these cultures aggregated to form three-dimensional structures called “attached ovarian epispheres”. The availability of these actively proliferating cell cultures allowed us to measure NER functionally and quantitatively by the unscheduled DNA synthesis (UDS) assay, a clinical test used to diagnose constitutive deficiencies in NER capacity. We determined that ovarian epithelial cells manifested an intermediate level of NER capacity in humans, viz., only 25% of that of foreskin fibroblasts, but still 2.5-fold higher than that of peripheral blood lymphocytes. This level of DNA repair capacity was indistinguishable from that of normal breast epithelial cells, suggesting that it might be characteristic of the epithelial cell type. Similar levels of NER activity were observed in cultures established from a disease-free known carrier of a BRCA1 truncation mutation, consistent with previous normal results shown in breast epithelium and blood lymphocytes. These results establish that at least three “normal” levels of such DNA repair occur in human tissues, and that NER capacity is epigenetically regulated during cell differentiation and development. PMID:18575893

Johnson, Jennifer M.; Miles, Tiffany D.; Dimsdale, Jason M.; Edwards, Robert P.; Kelley, Joseph L.; Grant, Stephen G.

2015-01-01

320

Differential marker expression by cultures rich in mesenchymal stem cells  

PubMed Central

Background Mesenchymal stem cells have properties that make them amenable to therapeutic use. However, the acceptance of mesenchymal stem cells in clinical practice requires standardized techniques for their specific isolation. To date, there are no conclusive marker (s) for the exclusive isolation of mesenchymal stem cells. Our aim was to identify markers differentially expressed between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. We compared and contrasted the phenotype of tissue cultures in which mesenchymal stem cells are rich and rare. By initially assessing mesenchymal stem cell differentiation, we established that bone marrow and breast adipose cultures are rich in mesenchymal stem cells while, in our hands, foreskin fibroblast and olfactory tissue cultures contain rare mesenchymal stem cells. In particular, olfactory tissue cells represent non-stem cell mesenchymal cells. Subsequently, the phenotype of the tissue cultures were thoroughly assessed using immuno-fluorescence, flow-cytometry, proteomics, antibody arrays and qPCR. Results Our analysis revealed that all tissue cultures, regardless of differentiation potential, demonstrated remarkably similar phenotypes. Importantly, it was also observed that common mesenchymal stem cell markers, and fibroblast-associated markers, do not discriminate between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. Examination and comparison of the phenotypes of mesenchymal stem cell and non-stem cell mesenchymal cell cultures revealed three differentially expressed markers – CD24, CD108 and CD40. Conclusion We indicate the importance of establishing differential marker expression between mesenchymal stem cells and non-stem cell mesenchymal cells in order to determine stem cell specific markers. PMID:24304471

2013-01-01

321

In Vitro Assays Using Primary Embryonic Mouse Lymphatic Endothelial Cells Uncover Key Roles for FGFR1 Signalling in Lymphangiogenesis  

PubMed Central

Despite the importance of blood vessels and lymphatic vessels during development and disease, the signalling pathways underpinning vessel construction remain poorly characterised. Primary mouse endothelial cells have traditionally proven difficult to culture and as a consequence, few assays have been developed to dissect gene function and signal transduction pathways in these cells ex vivo. Having established methodology for the purification, short-term culture and transfection of primary blood (BEC) and lymphatic (LEC) vascular endothelial cells isolated from embryonic mouse skin, we sought to optimise robust assays able to measure embryonic LEC proliferation, migration and three-dimensional tube forming ability in vitro. In the course of developing these assays using the pro-lymphangiogenic growth factors FGF2 and VEGF-C, we identified previously unrecognised roles for FGFR1 signalling in lymphangiogenesis. The small molecule FGF receptor tyrosine kinase inhibitor SU5402, but not inhibitors of VEGFR-2 (SU5416) or VEGFR-3 (MAZ51), inhibited FGF2 mediated LEC proliferation, demonstrating that FGF2 promotes proliferation directly via FGF receptors and independently of VEGF receptors in primary embryonic LEC. Further investigation revealed that FGFR1 was by far the predominant FGF receptor expressed by primary embryonic LEC and correspondingly, siRNA-mediated FGFR1 knockdown abrogated FGF2 mediated LEC proliferation. While FGF2 potently promoted LEC proliferation and migration, three dimensional tube formation assays revealed that VEGF-C primarily promoted LEC sprouting and elongation, illustrating that FGF2 and VEGF-C play distinct, cooperative roles in lymphatic vascular morphogenesis. These assays therefore provide useful tools able to dissect gene function in cellular events important for lymphangiogenesis and implicate FGFR1 as a key player in developmental lymphangiogenesis in vivo. PMID:22792354

Betterman, Kelly L.; Harvey, Natasha L.

2012-01-01

322

Density gradient electrophoresis of cultured human embryonic kidney cells  

NASA Technical Reports Server (NTRS)

Ground based confirmation of the electrophoretic heterogeneity of human embryonic kidney cell cultures, the general characterization of their electrophoretic migration, and observations on the general properties of cultures derived from electrophoretic subpopulations were studied. Cell migration in a density gradient electrophoresis column and cell electrophoretic mobility was determined. The mobility and heterogeneity of cultured human embryonic kidney cells with those of fixed rat erythrocytes as model test particle was compared. Electrophoretically separated cell subpopulations with respect to size, viability, and culture characteristics were examined.

Plank, L. D.; Kunze, M. E.; Giranda, V.; Todd, P. W.

1985-01-01

323

Replication of Chinese sacbrood virus in primary cell cultures of Asian honeybee (Apis cerana).  

PubMed

A primary cell culture system was established for the first time from embryonic tissues of Asian honeybee, Apis cerana, and used to trace the early infection process of Chinese sacbrood virus (CSBV), an iflavirus in the family Iflaviridae. A monolayer of epithelium-like cells of A. cerana, approximately 8-10 ?m in diameter, was grown in Kimura's insect medium at 28 °C within 3-4 days of setting up the cultures. Such cultured cells were inoculated with CSBV purified from infected larvae or pupae for 2 h. In electron and confocal micrographs, viral particles accumulated as filamentous or vesicular inclusions in the cytoplasm of infected cultured cells at 36 h post-inoculation (hpi). Real-time quantitative RT-PCR assay showed that the expression levels of four cistrons of CSBV in the cultured cells increased rapidly between 12 and 48 hpi. This newly established primary cell culture derived from A. cerana will be useful for further studies of infection caused by CSBV. PMID:25139546

Xia, Xiaocui; Mao, Qianzhou; Wang, Haitao; Zhou, Bingfeng; Wei, Taiyun

2014-12-01

324

[Isolation and culture of fetal bovine intestine-derived epithelial stem cells and the differentiation into hepatocyte-like cells].  

PubMed

Objective To establish the culture system of fetal bovine intestinal epithelial stem cells (IESCs) in vitro, identify specific markers of the cell lines and analyze the differentiation potential into hepatocyte-like cells. Methods IESCs were isolated from the 3- to 5-month fetal bovine intestine by the digestion of collagenase I, and cultured in the DMEM/F12 medium. The cell morphology was observed, and the proliferation ability and multiple differentiation potential were demonstrated by subculturing and its growth curve. The mRNA expressions of the surface markers Bmi1, Hes1, Lgr5 and cytokeratin 19 (CK19) were determined by reverse transcription PCR (RT-PCR), and the protein levels of Bmi1, LGR5 and CK19 were detected by immunofluorescence cytochemistry. Under the induction of fibroblast growth factor 4 (FGF-4) and hepatocyte growth factor (HGF), the cell differentiation into hepatocyte-like cells was assayed by the glycogen staining and RT-PCR. Results IESCs cultured in vitro expressed Bmi1, Hes1, Lgr5 and CK19 mRNAs, and CK19, Bmi1 and LGR5 proteins. The differentiated cells were positively stained by glycogen, and RT-PCR showed that the cells expressed ?-fetoprotein (AFP) and albumin (ALB) mRNAs. Conclusion The culture system of IESCs in vitro is successfully established, and the cells are differentiated into hepatocyte-like cells. PMID:25575060

Sun, Tingting; Cai, Lianshun; Guan, Weijun

2015-01-01

325

Cellulose Biosynthesis Inhibitors: Comparative Effect on Bean Cell Cultures  

PubMed Central

The variety of bioassays developed to evaluate different inhibition responses for cellulose biosynthesis inhibitors makes it difficult to compare the results obtained. This work aims (i) to test a single inhibitory assay for comparing active concentrations of a set of putative cellulose biosynthesis inhibitors and (ii) to characterize their effect on cell wall polysaccharides biosynthesis following a short-term exposure. For the first aim, dose-response curves for inhibition of dry-weight increase following a 30 days exposure of bean callus-cultured cells to these inhibitors were obtained. The compound concentration capable of inhibiting dry weight increase by 50% compared to control (I50) ranged from subnanomolar (CGA 325?615) to nanomolar (AE F150944, flupoxam, triazofenamide and oxaziclomefone) and micromolar (dichlobenil, quinclorac and compound 1) concentrations. In order to gain a better understanding of the effect of the putative inhibitors on cell wall polysaccharides biosynthesis, the [14C]glucose incorporation into cell wall fractions was determined after a 20 h exposure of cell suspensions to each inhibitor at their I50 value. All the inhibitors tested decreased glucose incorporation into cellulose with the exception of quinclorac, which increased it. In some herbicide treatments, reduction in the incorporation into cellulose was accompanied by an increase in the incorporation into other fractions. In order to appreciate the effect of the inhibitors on cell wall partitioning, a cluster and Principal Component Analysis (PCA) based on the relative contribution of [14C]glucose incorporation into the different cell wall fractions were performed, and three groups of compounds were identified. The first group included quinclorac, which increased glucose incorporation into cellulose; the second group consisted of compound 1, CGA 325?615, oxaziclomefone and AE F150944, which decreased the relative glucose incorporation into cellulose but increased it into tightly-bound cellulose fractions; and the third group, comprising flupoxam, triazofenamide and dichlobenil, decreased the relative glucose incorporation into cellulose and increased it into a pectin rich fraction. PMID:22489176

García-Angulo, Penélope; Alonso-Simón, Ana; Encina, Antonio; Álvarez, Jesús M.; Acebes, José L.

2012-01-01

326

Prescreening for environmental teratogens using cultured mesenchymal cells from the human embryonic palate  

SciTech Connect

Mesenchymal cells from the prefusion human embryonic palate have been established in culture and can be grown in either a serum-free hormone-supplemented medium or a serum-containing medium. The growth of these cells is quite rapid in culture and inhibited in a dose-dependent manner by most teratogens thus far tested, such as dexamethasone. These cells are highly sensitive to a variety of DNA synthetic and mitotic inhibitors. The responses of these cells are complementary to the ovarian tumor cell attachment assay of Braun et al (1, and in this volume). When used in conjunction with the tumor cells, the overall reliability is greater than 90% with only one false-negative, allopurinol.

Pratt, R.M.; Grove, R.I.; Willis, W.D.

1982-01-01

327

Effects of isobutyl 2-cyanoacrylate polymer on cultured cells derived from murine cerebral microvessels.  

PubMed

The initial and delayed effects of isobutyl 2-cyanoacrylate (IBCA) polymer on cells cultured from the wall of mouse cerebral microvessels were examined using a simple cytotoxicity assay. Cell cultured media were conditioned by exposure to IBCA polymer for 24-hr periods over the 15 days following polymerization and the media were then added to confluent endothelial or smooth muscle cell monolayers. The resulting cell detachment was assessed as a measure of cytotoxicity. The results indicated that immediately or a few days after polymerization and at approximately 12-13 days after polymerization, IBCA released material(s) that caused significant cell detachment from cell monolayers. This finding may explain some of the observed toxic effects of IBCA, particularly on vascular lesions within the brain. This simple bioassay may be used to study early and delayed effects of other potential embolotherapy materials on components of the blood vessel wall. PMID:20702356

Vinters, H V; Ho, H W

1988-01-01

328

Human Embryonic Stem Cells (hESCs) Cultured Under Distinctive Feeder-Free Culture Conditions Display Global Gene Expression Patterns Similar to hESCs from Feeder-Dependent Culture Conditions  

Microsoft Academic Search

Human embryonic stem cell (hESC)–based assay systems and genetically modified hESCs are very useful tools for screening drugs\\u000a that regulate stemness and differentiation and for studying the molecular mechanisms involved in hESC fate determination.\\u000a For these types of studies, feeder cell–dependent cultures of hESCs are often problematic because the physiology of the feeder\\u000a cells is perturbed by the drug treatments

Tae-Min Yoon; Bomi Chang; Hyeung-Taek Kim; Joo-Hyun Jee; Dong-Wook Kim; Dong-Youn Hwang

2010-01-01

329

Culture bag systems for clinical applications of adult human neural crest-derived stem cells  

PubMed Central

Introduction Facing the challenging treatment of neurodegenerative diseases as well as complex craniofacial injuries such as those common after cancer therapy, the field of regenerative medicine increasingly relies on stem cell transplantation strategies. Here, neural crest-derived stem cells (NCSCs) offer many promising applications, although scale up of clinical-grade processes prior to potential transplantations is currently limiting. In this study, we aimed to establish a clinical-grade, cost-reducing cultivation system for NCSCs isolated from the adult human nose using cGMP-grade Afc-FEP bags. Methods We cultivated human neural crest-derived stem cells from inferior turbinate (ITSCs) in a cell culture bag system using Afc-FEP bags in human blood plasma-supplemented medium. Investigations of viability, proliferation and expression profile of bag-cultured ITSCs were followed by DNA-content and telomerase activity determination. Cultivated ITSCs were introduced to directed in vitro differentiation assays to assess their potential for mesodermal and ectodermal differentiation. Mesodermal differentiation was determined using an enzyme activity assay (alkaline phosphatase, ALP), respective stainings (Alizarin Red S, Von Kossa and Oil Red O), and RT-PCR, while immunocytochemistry and synaptic vesicle recycling were applied to assay neuroectodermal differentiation of ITSCs. Results When cultivated within Afc-FEP bags, ITSCs grew three-dimensionally in a human blood plasma-derived matrix, thereby showing unchanged morphology, proliferation capability, viability and expression profile in comparison to three dimensionally-cultured ITSCs growing in standard cell culture plastics. Genetic stability of bag-cultured ITSCs was further accompanied by unchanged telomerase activity. Importantly, ITSCs retained their potential to differentiate into mesodermal cell types, particularly including ALP-active, Alizarin Red S-, and Von Kossa-positive osteogenic cell types, as well as adipocytes positive in Oil Red O assays. Bag culture further did not affect the potential of ITSCs to undergo differentiation into neuroectodermal cell types coexpressing ?-III-tubulin and MAP2 and exhibiting the capability for synaptic vesicle recycling. Conclusions Here, we report for the first time the successful cultivation of human NCSCs within cGMP-grade Afc-FEP bags using a human blood plasma-supplemented medium. Our findings particularly demonstrate the unchanged differentiation capability and genetic stability of the cultivated NCSCs, suggesting the great potential of this culture system for future medical applications in the field of regenerative medicine. PMID:24629140

2014-01-01

330

Effect of LLLT on endothelial cells culture.  

PubMed

Growth factors as vascular endothelial growth factor (VEGF), produced by the endothelial cells, take an essential part in pathological and physiological angiogenesis. The possibility of angiogenesis modulation by application of laser radiation may contribute to the improvement of its use in this process. Thus, the aim of the study was to investigate the influence of low-level laser therapy (LLLT) on the proliferation of endothelial cells, secretion of VEGF-A and presence of soluble VEGF receptors (sVEGFR-1 and sVEGFR-2) in the medium after in vitro culture. Isolated human umbilical vein endothelial cells (HUVECs) were irradiated using a diode laser at a wavelength of 635 nm and power density of 1,875 mW/cm(2). Depending on radiation energy density, the experiment was conducted in four groups: I 0 J/cm(2) (control group), II 2 J/cm(2), III 4 J/cm(2), and IV 8 J/cm(2). The use of laser radiation wavelength of 635 nm, was associated with a statistically significant increase in proliferation of endothelial cells (p?=?0.0041). Moreover, at 635-nm wavelength, all doses of radiation significantly reduced the concentration of sVEGFR-1 (p?=?0.0197). PMID:25231826

Góralczyk, Krzysztof; Szyma?ska, Justyna; ?ukowicz, Ma?gorzata; Drela, Ewelina; Kotzbach, Roman; Dubiel, Mariusz; Michalska, Ma?gorzata; Góralczyk, Barbara; Zaj?c, Andrzej; Ro??, Danuta

2015-01-01

331

Multiplex profiling of cellular invasion in 3D cell culture models.  

PubMed

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. PMID:23671660

Burgstaller, Gerald; Oehrle, Bettina; Koch, Ina; Lindner, Michael; Eickelberg, Oliver

2013-01-01

332

Characterisation of the membrane transport of pilocarpine in cell suspension cultures of Pilocarpus microphyllus.  

PubMed

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

Andreazza, Nathalia Luiza; Abreu, Ilka Nacif; Sawaya, Alexandra Christine Helena Frankland; Mazzafera, Paulo

2014-11-20

333

Ilex paraguariensis cell suspension culture characterization and response against ethanol  

Microsoft Academic Search

Cell suspension cultures of Ilex paraguariensis, a South American native tree known as the maté plant, were initiated in order to investigate plant defense. Cultures were characterized for their cell growth, chemical composition and sugar consumption. The present work quantified some effects of salicylic acid, methyl jasmonate, cellulase and ethanol on cell growth and sugar metabolism. Results suggest that salicylic

Kátia H. Kraemer; Eloir P. Schenkel; Robert Verpoorte

2002-01-01

334

Biolistic transformation of cotton embryogenic cell suspension cultures  

Technology Transfer Automated Retrieval System (TEKTRAN)

Genetic transformation of cotton is highly dependent on the ability to regenerate fertile plants from transgenic cells through somatic embryogenesis. Induction of embryogenic cell cultures is genotype-dependant. However, once embryogenic cell cultures are available, they can be effectively used fo...

335

Cholera toxin stimulation of human mammary epithelial cells in culture  

SciTech Connect

Addition of cholera toxin to human mammary epithelial cultures derived from reduction mammoplasties and primary carcinomas greatly stimulated cell growth and increased the number of times the cells could be successfully subcultured. Other agents known to increase intracellular cAMP levels were also growth stimulatory. The increased growth potential conferred by cholera toxin enhances the usefulness of this cell culture system.

Stampfer, M.R.

1982-06-01

336

Protective effect of salidroside against H 2 O 2 -induced cell apoptosis in primary culture of rat hippocampal neurons  

Microsoft Academic Search

Salidroside, a phenylpropanoid glycoside separated from a medicinal plant Rhodiola rosea, has been documented to have protective effects on neuronal cells in vitro. This study investigated whether salidroside was\\u000a able to extend its unique neuroprotection to primary cultured rat hippocampal neurons against hydrogen peroxide (H2O2)-induced cell damage. Cell viability tests and cell apoptosis assays confirmed that salidroside pretreatment attenuated H2O2-stimulated

Xia Chen; Qi Zhang; Qiong Cheng; Fei Ding

2009-01-01

337

A cell-based assay for aggregation inhibitors as therapeutics of polyglutamine-repeat disease and validation in Drosophila  

PubMed Central

The formation of polyglutamine-containing aggregates and inclusions are hallmarks of pathogenesis in Huntington's disease that can be recapitulated in model systems. Although the contribution of inclusions to pathogenesis is unclear, cell-based assays can be used to screen for chemical compounds that affect aggregation and may provide therapeutic benefit. We have developed inducible PC12 cell-culture models to screen for loss of visible aggregates. To test the validity of this approach, compounds that inhibit aggregation in the PC12 cell-based screen were tested in a Drosophila model of polyglutamine-repeat disease. The disruption of aggregation in PC12 cells strongly correlates with suppression of neuronal degeneration in Drosophila. Thus, the engineered PC12 cells coupled with the Drosophila model provide a rapid and effective method to screen and validate compounds. PMID:12730384

Apostol, Barbara L.; Kazantsev, Alexsey; Raffioni, Simona; Illes, Katalin; Pallos, Judit; Bodai, Laszlo; Slepko, Natalia; Bear, James E.; Gertler, Frank B.; Hersch, Steven; Housman, David E.; Marsh, J. Lawrence; Thompson, Leslie Michels

2003-01-01

338

Radioresistance of rat glioma cell lines cultured as multicellular spheroids. Correlation with electrical cell-to-cell-coupling.  

PubMed

Two chemically induced rat glioblastomas, RG2 and F98, were cultured as monolayers and as multicellular spheroids and subjected to Co-gamma-irradiation. In parallel, intercellular communication between cells was determined as electrical coupling between neighbouring cells using micro-electrode techniques. A third glioblastoma with known radiobiological response (9L) was assayed with respect to intercellular communication and included into this analysis. Electrical coupling was low for RG2, intermediate for F98, and high for 9L. Radioresistance of spheroids, as expressed in terms of the mean inactivation dose computed from the survival curves increased in the same direction (RG2: 2.4 Gy; F98: 5.1 Gy; 9L: 6.5 Gy). A comparison of these parameters demonstrates a correlation between solid tumor radioresistance and gap-junctional cell-to-cell communication, at least for the class of glioblastomas analysed in this study. PMID:2315846

Knedlitschek, G; Anderer, U; Weibezahn, K F; Dertinger, H

1990-02-01

339

A study of communication specificity between cells in culture  

PubMed Central

We have examined the specificity of communication between cells in culture by co-culturing cells derived from mammalian, avian, and arthropod organisms. Both mammalian and avian culture cells have similar gap junctional phenotypes, while the insect (arthropod) cell lines have a significantly different gap junctional structure. Electrophysiological and ultrastructural methods were used to examine ionic coupling and junctional interactions between homologous and heterologous cell types. In homologous cell systems, gap junctions and ionic coupling are present at a high incidence. Also, heterologous vertebrate cells in co-culture can communicate readily. By contrast, practically no coupling (0-8%) is detectable between heterologous insect cell lines (Homopteran or Lepidopteran) and vertebrate cells (mammalian myocardial or 3T3 cells). No gap junctions have been observed between arthropod and vertebrate cell types, even though the heterologous cells may be separated by less than 10 nm. In additional studies, a low incidence of coupling was found between heterologous insect cell lines derived from different arthropod orders. However, extensive coupling was detected between insect cell lines that are derived from the same order (Homoptera). These observations suggest that there is little or no apparent specificity for communication between vertebrate cells in culture that express the same gap junctional phenotype, while there is a definite communication specificity that exists between arthropod cells in culture. PMID:562887

1977-01-01

340

Clinical Impact of a PCR Assay for Identification of Staphylococcus aureus and Determination of Methicillin Resistance Directly from Blood Cultures  

PubMed Central

We evaluated the clinical usefulness of a PCR assay that discriminates Staphylococcus aureus from coagulase-negative staphylococci and detects methicillin resistance on blood cultures by measuring the adaptation of antimicrobial therapy based on the PCR results. Only 7 of 28 patients (25%) benefited from a modification of antibiotic therapy based on the PCR results, since empirical therapy was appropriate in a majority of cases. PMID:12904425

Hallin, M.; Maes, N.; Byl, B.; Jacobs, F.; De Gheldre, Y.; Struelens, M. J.

2003-01-01

341

Particle Trajectories in Rotating Wall Cell Culture Devices  

NASA Technical Reports Server (NTRS)

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.

Ramachandran N.; Downey, J. P.

1999-01-01

342

COMMUNICATION TO THE EDITOR High Cell Density Culture of  

E-print Network

COMMUNICATION TO THE EDITOR High Cell Density Culture of Metabolically Engineered Escherichia coli- tation processes employing several different bacteria have been developed to improve PHB productivity

343

Reduction of misleading ("false") positive results in mammalian cell genotoxicity assays. I. Choice of cell type.  

PubMed

Current in vitro mammalian cell genotoxicity assays show a high rate of positive results, many of which are misleading when compared with in vivo genotoxicity or rodent carcinogenicity data. P53-deficiency in many of the rodent cell lines may be a key factor in this poor predictivity. As part of an European Cosmetics Industry Association initiative for improvement of in vitro mammalian cell assays, we have compared several rodent cell lines (V79, CHL, CHO) with p53-competent human peripheral blood lymphocytes (HuLy), TK6 human lymphoblastoid cells, and the human liver cell line, HepG2. We have compared in vitro micronucleus (MN) induction following treatment with 19 compounds that were accepted as producing misleading or "false" positive results in in vitro mammalian cell assays [6]. Of these, six chemicals (2-ethyl-1,3-hexandiol, benzyl alcohol, urea, sodium saccharin, sulfisoxazole and isobutyraldehyde) were not toxic and did not induce any MN at concentrations up to 10mM. d,l-Menthol and ethionamide induced cytotoxicity, but did not induce MN. o-Anthranilic acid was not toxic and did not induce MN in V79, CHL, CHO, HuLy and HepG2 cells up to 10mM. Toxicity was induced in TK6 cells, although there were no increases in MN frequency up to and above the 55% toxicity level. The other 10 chemicals (1,3-dihydroxybenzene, curcumin, propyl gallate, p-nitrophenol, ethyl acrylate, eugenol, tert-butylhydroquinone, 2,4-dichlorophenol, sodium xylene sulfonate and phthalic anhydride) produced cytotoxicity in at least one cell type, and were evaluated further for MN induction in most or all of the cell types listed above. All these chemicals induced MN at concentrations <10mM, with levels of cytotoxicity below 60% (measured as the replication index) in at least one cell type. The rodent cell lines (V79, CHO and CHL) were consistently more susceptible to cytotoxicity and MN induction than p53-competent cells, and are therefore more susceptible to giving misleading positive results. These data suggest that a reduction in the frequency of misleading positive results can be achieved by careful selection of the mammalian cell type for genotoxicity testing. PMID:22138618

Fowler, Paul; Smith, Katie; Young, Jamie; Jeffrey, Laura; Kirkland, David; Pfuhler, Stefan; Carmichael, Paul

2012-02-18

344

mRNA Transfection of Mouse and Human Neural Stem Cell Cultures  

PubMed Central

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages. PMID:24386231

McLenachan, Samuel; Zhang, Dan; Palomo, Ana Belén Alvarez; Edel, Michael J.; Chen, Fred K.

2013-01-01

345

Microfluidic devices for cell culture and handling in organ-on-a-chip applications  

NASA Astrophysics Data System (ADS)

For many problems in system biology or pharmacology, in-vivo-like models of cell-cell interactions or organ functions are highly sought after. Conventional stationary cell culture in 2D plates quickly reaches its limitations with respect to an in-vivo like expression and function of individual cell types. Microfabrication technologies and microfluidics offer an attractive solution to these problems. The ability to generate flow as well as geometrical conditions for cell culture and manipulation close to the in-vivo situation allows for an improved design of experiments and the modeling of organ-like functionalities. Furthermore, reduced internal volumes lead to a reduction in reagent volumes necessary as well as an increased assay sensitivity. In this paper we present a range of microfluidic devices designed for the co-culturing of a variety of cells. The influence of substrate materials and surface chemistry on the cell morphology and viability for long-term cell culture has been investigated as well as strategies and medium supply for on-chip cell cultivation.

Becker, Holger; Schulz, Ingo; Mosig, Alexander; Jahn, Tobias; Gärtner, Claudia

2014-03-01

346

Morphology, proliferation, and osteogenic differentiation of mesenchymal stem cells cultured on titanium, tantalum, and chromium surfaces.  

PubMed

Metallic implants are widely used in orthopedic surgery and dentistry. Durable osseous fixation of an implant requires that osteoprogenitor cells attach and adhere to the implant, proliferate, differentiate into osteoblasts, and produce mineralized matrix. In the present study, we investigated the 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 days). Osteogenic differentiation response was quantified by cell-specific alkaline phosphatase activity (ALP) assay (4 days), expression analysis of bone-related genes (4 days), and mineralization assay (21 days). Undifferentiated and osteogenically stimulated MSCs cultured on the different surfaces showed the same tendencies for proliferation and differentiation. MSCs exposed to Ti surfaces demonstrated enhanced proliferation compared with Ta and Cr surfaces. Cultivation of MSCs on Ta surfaces resulted in significantly increased mean cellular area and cell-specific ALP activity compared with the 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. PMID:17975813

Stiehler, Maik; Lind, Martin; Mygind, Tina; Baatrup, Anette; Dolatshahi-Pirouz, Alireza; Li, Haisheng; Foss, Morten; Besenbacher, Flemming; Kassem, Moustapha; Bünger, Cody

2008-08-01

347

Mutations responsible for adaptation of hepatitis A virus to efficient growth in cell culture.  

PubMed Central

Chimeric genomes of hepatitis A virus strain HM-175 were constructed from cDNA clones of the wild-type virus and its cell culture-adapted variant. RNA transcribed in vitro from each construct was assayed for infectivity by transfection of cultured cells. RNA transcribed from the wild-type cDNA clone was minimally infectious and produced virus that grew inefficiently in vitro, whereas that transcribed from certain chimeric genomes consistently produced virus that grew efficiently in cultured cells. Mutations in the P2 region were found to be necessary for efficient virus growth in vitro, while mutations in the 5' noncoding region imparted a conditional enhancement of growth in vitro. Images PMID:1651411

Emerson, S U; McRill, C; Rosenblum, B; Feinstone, S; Purcell, R H

1991-01-01

348

Demonstration of toxicity to fish and to mammalian cells by Pfiesteria species: Comparison of assay methods and strains  

PubMed Central

Toxicity and its detection in the dinoflagellate fish predators Pfiesteria piscicida and Pfiesteria shumwayae depend on the strain and the use of reliable assays. Two assays, standardized fish bioassays (SFBs) with juvenile fish and fish microassays (FMAs) with larval fish, were compared for their utility to detect toxic Pfiesteria. The comparison included strains with confirmed toxicity, negative controls (noninducible Pfiesteria strains and a related nontoxic cryptoperidiniopsoid dinoflagellate), and P. shumwayae strain CCMP2089, which previously had been reported as nontoxic. SFBs, standardized by using toxic Pfiesteria (coupled with tests confirming Pfiesteria toxin) and conditions conducive to toxicity expression, reliably detected actively toxic Pfiesteria, but FMAs did not. Pfiesteria toxin was found in fish- and algae-fed clonal Pfiesteria cultures, including CCMP2089, but not in controls. In contrast, noninducible Pfiesteria and cryptoperidiniopsoids caused no juvenile fish mortality in SFBs even at high densities, and low larval fish mortality by physical attack in FMAs. Filtrate from toxic strains of Pfiesteria spp. in bacteria-free media was cytotoxic. Toxicity was enhanced by bacteria and other prey, especially live fish. Purified Pfiesteria toxin extract adversely affected mammalian cells as well as fish, and it caused fish death at environmentally relevant cell densities. These data show the importance of testing multiple strains when assessing the potential for toxicity at the genus or species level, using appropriate culturing techniques and assays. PMID:15728353

Burkholder, JoAnn M.; Gordon, Andrew S.; Moeller, Peter D.; Law, J. Mac; Coyne, Kathryn J.; Lewitus, Alan J.; Ramsdell, John S.; Marshall, Harold G.; Deamer, Nora J.; Cary, S. Craig; Kempton, Jason W.; Morton, Steven L.; Rublee, Parke A.

2005-01-01

349

Skeletal muscle satellite cells cultured in simulated microgravity  

NASA Technical Reports Server (NTRS)

Satellite cells are postnatal myoblasts responsible for providing additional nuclei to growing or regenerating muscle cells. Satellite cells retain the capacity to proliferate and differentiate in vitro and therefore provide a useful model to study postnatal muscle development. Most culture systems used to study postnatal muscle development are limited by the two-dimensional (2-D) confines of the culture dish. Limiting proliferation and differentiation of satellite cells in 2-D could potentially limit cell-cell contacts important for developing the level of organization in skeletal muscle obtained in vivo. Culturing satellite cells on microcarrier beads suspended in the High-Aspect-Ratio-Vessel (HARV) designed by NASA provides a low shear, three-dimensional (3-D) environment to study muscle development. Primary cultures established from anterior tibialis muscles of growing rats (approximately 200 gm) were used for all studies and were composed of greater than 75 % satellite cells. Different inoculation densities did not affect the proliferative potential of satellite cells in the HARV. Plating efficiency, proliferation, and glucose utilization were compared between 2-D flat culture and 3-D HARV culture. Plating efficiency (cells attached - cells plated x 100) was similar between the two culture systems. Proliferation was reduced in HARV cultures and this reduction was apparent for both satellite cells and non-satellite cells. Furthermore, reduction in proliferation within the HARV could not be attributed to reduced substrate availability since glucose levels in media from HARV and 2-D cell culture were similar. Morphologically, microcarrier beads within the HARVS were joined together by cells into three-dimensional aggregates composed of greater than 10 beads/aggregate. Aggregation of beads did not occur in the absence of cells. Myotubes were often seen on individual beads or spanning the surface of two beads. In summary, proliferation and differentiation of satellite cells on microcarrier beads within the HARV bioreactor results in a three dimensional level of organization that could provide a more suitable model to study postnatal muscle development.

Molnar, Greg; Hartzell, Charles R.; Schroedl, Nancy A.; Gonda, Steve R.

1993-01-01

350

Bovine recto-anal junction squamous epithelial (RSE) cell adhesion assay for studying Escherichia coli O157 adherence  

Technology Transfer Automated Retrieval System (TEKTRAN)

An adherence assay, using recto-anal junction squamous epithelial cells (RSEC), was developed for Escherichia coli O157 and related organisms. The assay was standardized in comparison with the routinely used HEp-2 cell adherence assay, in this “proof of concept” study. The novel RSEC adhesion assay ...

351

Three-dimensional tissue culture based on magnetic cell levitation  

NASA Astrophysics Data System (ADS)

Cell culture is an essential tool in drug discovery, tissue engineering and stem cell research. Conventional tissue culture produces two-dimensional cell growth with gene expression, signalling and morphology that can be different from those found in vivo, and this compromises its clinical relevance. Here, we report a three-dimensional tissue culture based on magnetic levitation of cells in the presence of a hydrogel consisting of gold, magnetic iron oxide nanoparticles and filamentous bacteriophage. By spatially controlling the magnetic field, the geometry of the cell mass can be manipulated, and multicellular clustering of different cell types in co-culture can be achieved. Magnetically levitated human glioblastoma cells showed similar protein expression profiles to those observed in human tumour xenografts. Taken together, these results indicate that levitated three-dimensional culture with magnetized phage-based hydrogels more closely recapitulates in vivo protein expression and may be more feasible for long-term multicellular studies.

Souza, Glauco R.; Molina, Jennifer R.; Raphael, Robert M.; Ozawa, Michael G.; Stark, Daniel J.; Levin, Carly S.; Bronk, Lawrence F.; Ananta, Jeyarama S.; Mandelin, Jami; Georgescu, Maria-Magdalena; Bankson, James A.; Gelovani, Juri G.; Killian, T. C.; Arap, Wadih; Pasqualini, Renata

2010-04-01

352

Cell and tissue culture of Miscanthus Sacchariflorus  

SciTech Connect

Since recent time search and introduction of new species of plants have paid attention. More perspective are perennial low maintenance landscape plants from genera Phragmites L. and Miscanthus Anderss. known as high speed growing and great amount of cellulose`s containing. Absence of seeds production and limited distribution area prevent from immediately introduction the plants of this species. The main goal of our investigation is the scientific development of the cell and tissue culture methods to get changing clones, salt and cold tolerant plants and their micropogation. At present there are collection of biovariety represented by subspecies, ecotypes and plant regenerants of two species - Miscanthus purpurascens (Anders.) and Miscanthus sacchariflorus (Maxim.). Successful results have been achieved in screening of culture media, prepared on MS base medium and contained a row of tropic components to protect the explant and callus tissue from oxidation and necrosis. Initially the callus was induced from stem segments, apical and nodular meristem of vegetative shoots of elulalia, growing in hydroponic greenhouse. Morphological and cytologic analysis of plant-regenerants have been done.

Godovikova, V.A.; Moiseyeva, E.A.; Shumny, V.K. [Institute of Cytology and Genetics, Novosibirsk (Russian Federation)

1995-11-01

353

Free radical damage to cultured porcine aortic endothelial cells and lung fibroblasts: Modulation by culture conditions  

Microsoft Academic Search

Summary  Culture conditions modulating cell damage from xanthine plus xanthine oxidase-derived partially reduced oxygen species were\\u000a studied. Porcine thoracic aorta endothelial cells and porcine lung fibroblasts were maintained in monolayer culture. Cells\\u000a were prelabeled with51Cr before xanthine plus xanthine oxidase exposure. Endothelial cells showed 30 to 100% more lysis than fibroblasts and thus\\u000a seemed more sensitive to this oxidant stress. The

Clark T. Bishop; Zermeena Mirza; James D. Crapo; Bruce A. Freeman

1985-01-01

354

A Cell Lysis and Protein Purification - Single Molecule Assay Devices for Evaluation of Genetically Engineered Proteins  

NASA Astrophysics Data System (ADS)

We have developed two devices applicable to evaluate genetically engineered proteins in single molecule assay: on-chip cell lysis device, and protein purification - assay device. A motor protein, F1-ATPase expressed in E.coli, was focused in this report as a target protein. Cell lysis was simply performed by applying pulse voltage between Au electrodes patterned by photolithography, and its efficiency was determined by absorptiometry. The subsequent processes, purification and assay of extracted proteins, were demonstrated in order to detect F1-ATPase and to evaluate its activity. The specific bonding between his-tag in F1-ATPase and Ni-NTA coated on a glass surface was utilized for the purification process. After immobilization of F1-ATPase, avidin-coated microspheres and adenosine tri-phosphate (ATP) solution were infused sequentially to assay the protein. Microsphere rotation was realized by activity of F1-ATPase corresponding to ATP hydrolysis. Results show that the cell lysis device, at the optimum condition, extracts enough amount of protein for single molecule assay. Once cell lysate was injected to the purification - assay device, proteins were diffused in the lateral direction in a Y-shape microchannel. The gradient of protein concentratioin provides an optimal concentration for the assay i.e. the highest density of rotating beads. Density of rotating beads is also affected by the initial concentration of protein injected to the device. The optimum concentration was achieved by our cell lysis device not by the conventional method by ultrasonic wave. Rotation speed was analyzed for several microspheres assayed in the purification - assay device, and the results were compatible to that of conventional assay in which F1-ATPase was purified in bulk scale. In conclusion, we have demonstrated on-chip cell lysis and assay appropriate for the sequential analysis without any pretreatment. On-chip devices replacing conventional bioanalytical methods will be integrated a total analysis system to evaluate engineered protein and DNA.

Nakyama, Tetsuya; Tabata, Kazuhito; Noji, Hiroyuki; Yokokawa, Ryuji

355

Bovine mammary epithelial cells retain stem-like phenotype in long-term cultures.  

PubMed

The detection and characterization of bovine mammary stem cells may give a better understanding of the cyclic characteristic of mammary gland development. In turn, this could potentially offer techniques to manipulate lactation yield and for regenerative medicine. We previously demonstrated that adult stem cells reside in the bovine mammary gland and possess an intrinsic regenerative potential. In vitro maintenance and expansion of this primitive population is a challenging task that could make easier the study of adult mammary stem cells. The aim of this study is to investigate this possibility. Different subpopulations of mammary epithelial cells emerge when they are cultured in two defined culture conditions. Specific cell differentiation markers as cytokeratin 18 (CK18) and cytokeratin 14 (CK14) were expressed with significant differences according to culture conditions. Vimentin, a well-known fibroblast marker was observed to increase significantly (P?culture (25 days) a subset of cells still retained regenerative capabilities. These cells were able to form organized pseudo-alveoli when transplanted in immunodeficient mice as shown by the expression of cytokeratin 14 (CK14), cytokeratin 18 (CK18), p63 (a mammary basal cell layer marker) and Epithelial Cell Adhesion Molecule (EpCAM). We also were able to observe the presence of milk proteins signal in these regenerated structures, which is a specific marker of functional mammary alveoli. Progenitor content was also analyzed in vitro through Colony-Forming Cell (CFC) assays with no substantial differences among culture conditions and time points. These results demonstrate that long-term culture of a multipotent cell subpopulation with intrinsic regenerative potential is possible. PMID:25189469

Diego, Cravero; Eugenio, Martignani; Silvia, Miretti; Elisabetta, Macchi; Paolo, Accornero; Mario, Baratta

2014-10-01

356

In vitro tests of resveratrol radiomodifying effect on rhabdomyosarcoma cells by comet assay.  

PubMed

Cancer is a global public health problem. Resveratrol is a defensive polyphenol that is synthesized by a wide variety of plants in response to exposure to ultraviolet radiation or also due to mechanical stress caused by the action of pathogens and chemical and physical agents. Grape vines have a high capacity to produce resveratrol, so grape juice and wine, mainly red wine, are considered good sources of resveratrol. The protective effects of resveratrol include promotion of antiinflammatory response, antitumor activity and prevention of degenerative diseases, reduced incidence of cardiovascular diseases and inhibition of platelet aggregation, among others. Therefore, resveratrol is considered to be a cell protector. However, at high concentrations, resveratrol promotes contrary effects by sensitizing cells. The aim of this study was to investigate in vitro the radiomodifying effect of resveratrol in culture of human rhabdomyosarcoma cells (RD) by applying the comet assay to evaluate the cell damage and repair capacity. The LD50 (lethal dose) obtained was 499.95 ± 9.83 Gy (Mean ± SD) and the CI50 (cytotoxicity index) was 150 ?M in the RD cells. Based on these data, it was defined the gamma radiation doses (50 and 100 Gy) and resveratrol concentrations (15, 30 and 60 ?M) to be used in this study. The results indicated that resveratrol acts as a cell protector at a concentration of 15 ?M and has a cytotoxic effect at 60 ?M. However, with the interaction of the gamma radiation, the concentration of 60 ?M did not produce a statistically significant radiosensitizing effect. PMID:25084316

Magalhães, V D; Rogero, S O; Cruz, A S; Vieira, D P; Okazaki, K; Rogero, J R

2014-12-01

357

Cytotoxicity of Trichoderma spp. cultural filtrate against human cervical and breast cancer cell lines.  

PubMed

Trichoderma spp. are known as a rich source of secondary metabolites with biological activity belonging to a variety of classes of chemical compounds. These fungi also are well known for their ability to produce a wide range of antibiotic substances and to parasitize other fungi. In search for new substances, which might act as anticancer agents, the overall objective of this study was to investigate the cytotoxic effects of Trichoderma harzianum and Trichoderma asperellum cultural filtrates against human cervical and breast cancer cell lines (HeLa and MCF-7 cells respectively). To achieve this objective, cells were exposed to 20, 40, 60, 80 and 100 mg/ ml of both T. harzianum cultural filtrate (ThCF) and T. asperellum cultural filtrate (TaCF) for 24h, then the cell viability and the cytotoxic responses were assessed by using trypan blue and 3-(4,5-dimethylthiazol-2yl)- 2,5-biphenyl tetrazolium bromide (MTT) assays. Morphological changes in cells were investigated by phase contrast inverted microscopy. The results showed that ThCF and TaCF significantly reduce the cell viability, have cytotoxic effects and alter the cellular morphology of HeLa and MCF-7 cells in a concentration dependent manner. A concentration of 80 and 100mg/ml of ThCF resulted in a sharp decline in the cell viability percent of HeLa and MCF-7 respectively (25.2%, 26.5%) which was recorded by trypan blue assay. The half-maximal inhibitory concentrations (IC50) of ThCF and TaCF in HeLa and MCF-7 were recorded as 16.6, 12.0, 19.6 and 0.70 mg/ml respectively by MTT assay. These results revealed that ThCF and TaCF have a substantial ability to reduce the viability and proliferation of human cervical and breast cancer cells. PMID:25227819

Abd El-Rahman, Atef Abd El-Mohsen; El-Shafei, Sally Mohamed Abd El-Aziz; Ivanova, Elena Vladimirovna; Fattakhova, Alfia Nurlimanovna; Pankova, Anna Victorovna; El-Shafei, Mohamed Abd El-Aziz; El-Morsi, El-Morsi Abu El-Fotouh; Alimova, Farida Kashifovna

2014-01-01

358

A spectrophotometer-based diffusivity assay reveals that diffusion hindrance of small molecules in extracellular matrix gels used in 3D cultures is dominated by viscous effects.  

PubMed

The design of 3D culture studies remains challenging due to the limited understanding of extracellular matrix (ECM)-dependent hindered diffusion and the lack of simple diffusivity assays. To address these limitations, we set up a cost-effective diffusivity assay based on a Transwell plate and the spectrophotometer of a Microplate Reader, which are readily accessible to cell biology groups. The spectrophotometer-based assay was used to assess the apparent diffusivity D of FITC-dextrans with molecular weight (4-70kDa) spanning the physiological range of signaling factors in a panel of acellular ECM gels including Matrigel, fibrin and type I collagen. Despite their technical differences, D data exhibited ?15% relative difference with respect to FRAP measurements. Our results revealed that diffusion hindrance of small particles is controlled by the enhanced viscosity of the ECM gel in conformance with the Stokes-Einstein equation rather than by geometrical factors. Moreover, we provided a strong rationale that the enhanced ECM viscosity is largely contributed to by unassembled ECM macromolecules. We also reported that gels with the lowest D exhibited diffusion hindrance closest to the large physiologic hindrance of brain tissue, which has a typical pore size much smaller than ECM gels. Conversely, sparse gels (?1mg/ml), which are extensively used in 3D cultures, failed to reproduce the hindered diffusion of tissues, thereby supporting that dense (but not sparse) ECM gels are suitable tissue surrogates in terms of macromolecular transport. Finally, the consequences of reduced diffusivity in terms of optimizing the design of 3D culture experiments were addressed in detail. PMID:24916283

Galgoczy, Roland; Pastor, Isabel; Colom, Adai; Giménez, Alicia; Mas, Francesc; Alcaraz, Jordi

2014-08-01

359

Establishment of a cloned line of Lewis Lung Carcinoma cells adapted to cell culture.  

PubMed

A cloned line of cells adapted to culture has been isolated from the Lewis Lung Carcinoma and has been designated the Lewis lung carcinoma line 1 (LLC1). It grows as a monolayer culture in RPMI 1640 medium supplemented with 2% fetal calf serum with a plating efficiency of about 94% and a doubling time of 21 h. LLC1 cells remain highly tumorigenic in C57B1 mice and produce primary tumors and lung metastases histologically indistinguishable from the original tumor line. The doubling time for a subcutaneous tumor derived from LLC1 cells was 23 h for a tumor mass of about 0.1 g and 40 h for a tumor mass of about 1 g. The cell line forms discrete colonies on a plastic substrate and can be used in a focus assay to determine drug induced cytotoxicity. Results with a number of chemotherapeutic agents are reported; in general, sensitivity measured in vitro does not correspond with published reports of sensitivity of the Lewis Lung carcinoma in vivo. PMID:7226139

Bertram, J S; Janik, P

1980-11-01

360

Establishment and characterization of a differentiated epithelial cell culture model derived from the porcine cervix uteri  

PubMed Central

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. PMID:22429795

2012-01-01

361

Prelining of polytetrafluoroethylene grafts with cultured human endothelial cells isolated from varicose veins.  

PubMed

Prelining graft material with autologous functioning endothelial cells might be one of the ultimate requirements to obtain a biocompatible surface. Accordingly, endothelial cells from stripped varicose veins were enzymatically harvested and grown on a fibronectin matrix. Proliferation was investigated in defined medium supplemented with various concentrations of endothelial cell growth supplement (ECGS) (25, up to 150 micrograms/ml) and heparin (10(-8), up to 10(-5)mol/L): optimal growth required both 150 micrograms/ml of ECGS and 10(-5)mol/L heparin. Under these conditions, cell culture achieved cell densities at a confluence of 1.2 +/- 1.1 10(5) cells/cm2 with a doubling time of 1 day. During subcultivation cultured cells consistently exhibited characteristic cobblestone morphology and immunofluorescent staining for factor VIII-related antigen, whereas prostacyclin production determined by enzyme-linked immunosorbent assay for 6-keto-prostaglandin F1 alpha reached 21.1 +/- 1.2 ng/10(6) cells after 15-minute stimulation with 1 U/ml of thrombin. Heparin-containing culture medium-endothelial cell interactions were particularly studied, and with iodine 125-heparin, binding was demonstrated with an apparent dissociation constant (Kd) of 0.36 +/- 0.04 mumol/L. A cold storage technique at -80 degrees C was sought, and freezed cells were used to coat in vitro polytetrafluoroethylene grafts. Protein-treated material allowed cell attachment and growth to a confluent monolayer as assayed by light and scanning electron microscopy. These data validate the feasibility of prelining grafts in vitro with autologous functioning endothelial cells. This approach may be useful in improving the performance of small-caliber vascular grafts according to prostacyclin production and surface-bound heparin of these cells. PMID:2643196

Leseche, G; Bikfalvi, A; Dupuy, E; Tobelem, G; Andreassian, B; Caen, J

1989-01-01

362

The influence of visible light exposure on cultured RGC-5 cells  

PubMed Central

Purpose To determine the effects of visible light on normal or metabolically compromised cultured rat RGC-5 cells. Methods Cultured RGC-5 cells were exposed to different durations as well as intensities of optical radiation, filtered to exclude wavelengths below 400 nm. Some cells were also subjected to metabolic compromise by depriving them of serum (serum deprivation; SD). Treated cells were assayed for cell viability using the 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, for DNA breakdown by terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-linked nick end labeling (TUNEL), apoptotic protein activation by immunoblotting, and the production of reactive oxygen species (ROS) with dihydroethidium. A subset of cells was treated with 100 pM rotenone as an alternative means to induce metabolic stress; this was to determine that the influence of light on compromised cells was not specific to serum-deprivation alone. Results Exposure to the light for 48 h activated both caspase-3 and Bcl-associated X-protein (Bax) in cultured RGC-5 cells. Furthermore, light (1000 or 4000 lux), SD, and rotenone caused minor but significant decreases in cellular MTT reduction. SD and light also led to cellular DNA breakdown, although only light caused ROS production. Light (48 h) significantly exacerbated the effect of SD on MTT reduction and DNA cleavage. Furthermore, the antioxidant, trolox, significantly blunted the detrimental influence of light on cell viability, increase in TUNEL-positive cells, and the generation of ROS. Conclusions Exposure to light was slightly, but significantly, harmful to healthy RGC-5 cells alone, but was much more toxic to those cells that were energetically compromised. Continuous light exposure can therefore detrimentally affect metabolically stressed RGC-5 cells. This may have implications for some ocular retinopathies such as glaucoma PMID:18334950

Wood, John P. M.; Lascaratos, Gerassimos; Bron, Anthony J.

2008-01-01

363

Culture of cells from mammalian tissue cryopreserved without cryoprotection  

E-print Network

Donor cells for nuclear transfer are usually prepared by the culture of fresh tissue. However, animal carcasses are sometimes frozen without cryoprotectants and if it were possible to obtain live cells from carcasses (tissue) preserved...

Charles, Lara Nicole

2009-05-15

364

Hepatotoxicity studies with primary cultures of rat liver cells  

Microsoft Academic Search

Summary  A method for preparing primary monolayer cultures of postnatal rat hepatocytes has been developed in our laboratory. Growing\\u000a cultures in arginine-deficient medium inhibits fibroblast overgrowth, and relatively pure cultures of parenchymal hepatocytes\\u000a are obtained. This cell culture system has been used to study the cytotoxicity of two hepatotoxic agents, tetracycline and\\u000a norethindrone. Caffeine was evaluated as an agent thought to

David C. Anuforo; Daniel Acosta; Robert V. Smith

1978-01-01

365

Three-dimensional cell culturing by magnetic levitation for evaluating efficacy/toxicity of photodynamic therapy  

NASA Astrophysics Data System (ADS)

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, 6.25?g/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 ?g/ml) is more aggressive compared to the effect of PDT in Hep G2 cells (EC50 = 7.48 ?g/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.

Sabino, Luis G.; Menezes, Priscila F. C.; Bagnato, Vanderlei S.; Souza, Glauco; Killian, Thomas C.; Kurachi, Cristina

2014-03-01

366

Imaging and Analysis of Three-Dimensional Cell Culture Models  

PubMed Central

Three-dimensional (3D) cell cultures are important tools in cell biology research and tissue engineering because they more closely resemble the architectural microenvironment of natural tissue, compared to standard two-dimensional cultures. Microscopy techniques that function well for thin, optically transparent cultures, however, are poorly suited for imaging 3D cell cultures. Three-dimensional cultures may be thick and highly scattering, preventing light from penetrating without significant distortion. Techniques that can image thicker biological specimens at high resolution include confocal microscopy, multiphoton microscopy, and optical coherence tomography. In this chapter, these three imaging modalities are described and demonstrated in the assessment of functional and structural features of 3D chitosin scaffolds, 3D micro-topographic substrates from poly-dimethyl siloxane molds, and 3D Matrigel cultures. Using these techniques, dynamic changes to cells in 3D microenvironments can be non-destructively assessed repeatedly over time. PMID:19957133

Graf, Benedikt W.; Boppart, Stephen A.

2013-01-01

367

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

PubMed Central

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

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

2014-01-01

368

Liposomes and MTT cell viability assay: An incompatible affair.  

PubMed

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is commonly used to evaluate the cytotoxicity potential of drugs vehicled by liposomes. However, liposome delivering drugs could produce inconsistent values of MTT absorbance. On the basis of previous experiments demonstrating the MTT affinity for lipid droplets, this paper aims to show that empty-liposomes interfere, per se, on MTT assay due to its lipidic nature. This brings into question the use of MTT testing cytotoxicity when liposomes are involved in delivering drugs. PMID:25481524

Angius, Fabrizio; Floris, Alice

2015-03-01

369

Effects of pantothenic acid on fibroblastic cell cultures  

Microsoft Academic Search

Summary To evaluate the effects of pantothenic acid during wound healing processes, fibroblastic cell cultures originating from foreskin were established and subcultured by trypsinization. PA (40 µg\\/ml) was added to the basal culture medium. The cell proliferation was estimated by cell count and determination of3H-thymidine incorporation. The protein synthesis and secretion were determined by dosage in the cells and in

B. Lacroix; E. Didier; J. F. Grenier

1988-01-01

370

Cerebral microvessels and derived cells in tissue culture  

Microsoft Academic Search

Summary  An endothelial cell line has been established from a primary culture of cerebral microvessels isolated from Swiss-Webster\\u000a mice. The microvessels were isolated by a mechanical dispersion and filtration technique. The cells that emerged from these\\u000a microvessels, maintained in organoid cultures, proliferated and formed plaques of a single or mixed cell type. The endothelial\\u000a cell line, designated ME-2, was isolated from

Lawrence E. DeBault; Eduardo Henriquez; Michael N. Hart; Pasquale A. Cancilla

1981-01-01

371

Dog pancreatic duct epithelial cells: long-term culture and characterization.  

PubMed Central

Epithelial cells, isolated from a normal dog pancreatic duct, were grown on collagen-coated culture inserts suspended above a feeder layer of myofibroblasts. The cells were examined by transmission electron microscopy, immunohistochemistry, cytogenetics, and flow cytometry. In addition, the constitutive and agonist-stimulated mucin secretion of these cells was studied using a [3H]N-acetyl-D-glucosamine labeling assay, and the stimulation of intracellular cAMP was measured. Cells grown on inserts with a feeder layer developed into confluent monolayers consisting of strictly polarized columnar epithelial cells with prominent microvilli, intercellular junctions, and normal chromosomal characteristics. They could be passaged repeatedly without a detectable alteration in their morphology. The cells could also be grown on organotypic cultures, resulting in further differentiated cells simulating in vivo morphology. Immunohistochemistry demonstrated the presence of carbonic anhydrase II in these cells. Cells treated with vasoactive intestinal peptide, epinephrine, and dibutyryl-cAMP demonstrated a marked increase in mucin secretion compared with controls. In parallel experiments, VIP and epinephrine significantly increased intracellular cAMP. In conclusion we have developed a pancreatic epithelial cell preparation with morphology, cytokinetics, chromosomal, and DNA analyses characteristic of normal cells. Similar to normal columnar epithelial cells, these pancreatic duct cells secreted mucin constitutively and responded to agonist by increasing secretion via a cAMP-mediated pathway. They also contained carbonic anhydrase, which indicates that the cells are capable of secreting bicarbonate. Images Figure 1 Figure 2 Figure 3 PMID:8774152

Oda, D.; Savard, C. E.; Nguyen, T. D.; Eng, L.; Swenson, E. R.; Lee, S. P.

1996-01-01

372

Psoriatic hair follicle cells. IV. Calmodulin levels in freshly isolated and cultured human scalp hair follicle cells.  

PubMed

Calmodulin levels were measured by radioimmuno-assay in freshly isolated and cultured psoriatic human scalp hair follicle cells. The mean value +/- SEM for calmodulin was 1.97 +/- 0.15 ng calmodulin micrograms-1 protein for 16 control subjects whereas calmodulin levels were significantly increased in psoriatic hair follicles, 2.93 +/- 0.26 ng calmodulin micrograms-1 protein (uninvolved skin) for 18 patients and 3.09 +/- 0.21 ng calmodulin micrograms-1 protein for involved skin derived hair follicles for 17 of these patients. In vitro, 3-week-old cultures of psoriatic keratinocytes contained less DNA and more calmodulin per DNA than their normal counterparts. When 6 week-old cultures of psoriatic and control hair follicle keratinocytes were compared, this difference disappeared. These results are related to the state of differentiation of these cultures. PMID:3807903

Lenoir, M C; Vromans, E; Shroot, B; Vermorken, A J

1986-01-01

373

Cell-line Engineering of Chinese Hamster Ovary Cells for Low-temperature Culture  

E-print Network

Developments in mammalian cell culture and recombinant technology has allowed for the production of recombinant proteins for use as human therapeutics. Mammalian cell culture is typically operated at the physiological ...

Kiat, Tan Hong

374

Co-culture of hepatocellular carcinoma cells and human umbilical endothelial cells damaged by SU11274  

PubMed Central

Mesenchymal-epithelial transition factor (c-Met) is a receptor that binds to the hepatocyte growth factor and is upregulated in hepatocellular carcinoma (HCC). The anti-tumor effects of (3Z)-N-(3-chlorophenyl)-3-({3,5-dimethyl-4-[(4- methyl-piperazin-1-yl)carbonyl]-1H-pyrrol-2-yl}methylene)-N-me- thyl-2-oxo-2,3-dihydro-1H-indole-5-sulfonamide (SU11274), a c-Met inhibitor, were investigated in the present study. HCC cells (HLE, HLF, PLC/PRL/5, Hep3B, Huh-6 and HepG2) and human umbilical vein endothelial cells (HUVECs) were used. Quantitative polymerase chain reaction was performed to detect the expression level of c-Met in HCC and HUVECs, and cyclin D1 in HCC. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-car-boxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt assay was performed to assess the proliferation of the HCC cells and HUVECs cultured with SU11274. Co-culture of HLF or PLC/PRL/5 cells and HUVECs was established as an in vitro model of HCC tissues. The expression levels of c-Met in HLE, HLF, PLC/PRL/5, Hep3B, Huh-6 and HepG2, adult healthy liver and HUVECs were 4.43±0.50, 1.61±0.18, 3.70±0.08, 0.81±0.18, 6.60±1.29, 1.06±0.35, 1.00±0.09 and 88.8±17.3 (mean ± standard deviation), respectively. SU11274 (30 ?M) suppressed the proliferation of HLF, PLC/PRL/5 and HUVECs to 11.0±9.4, 46.5±30.7 and 29.4±5.0%, respectively. SU11274 (30 ?M) decreased the expression levels of cyclin D1 in HLF and PLC/PRL/5 cells to 45.1±11.6 and 30.1±10.3%, respectively. SU11274, at a concentration of 30 ?M damaged the morphology of the co-cultures of HLF or PLC/PRL/5 cells with HUVECs and all the cells died. c-Met is highly expressed in HUVECs and HCC cells, but not in Hep3B. At a 30-?M concentration, SU11274 suppresses the proliferation of HLF, PLC/PRL/5 and HUVECs. SU11274 (30 ?M) damages the co-cultures of HLF or PLC/PRL/5 cells with HUVECs. PMID:25279148

TOMIZAWA, MINORU; SHINOZAKI, FUMINOBU; MOTOYOSHI, YASUFUMI; SUGIYAMA, TAKAO; YAMAMOTO, SHIGENORI; ISHIGE, NAOKI

2014-01-01

375

Detrimental effect of fast neutrons on cultured immature rat hippocampal cells: relative biological effectiveness of in vitro cell death indices.  

PubMed

This in vitro study compared the detrimental effect and relative biological effectiveness (RBE) of high-linear energy transfer (LET) fast neutrons on rat immature hippocampal cultured cells with those of low-LET ? rays. Immature hippocampal cells were exposed to fast neutrons or ? rays. Cytotoxicity and cell viability were analyzed using a lactate dehydrogenase (LDH)-release assay and a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, respectively. The cytotoxicity and cell viability with fast neutrons or ? rays varied in a dose-dependent pattern. In the LDH release and MTT assay indices, the RBEs of fast neutrons were approximately 2.35 and 2.42, respectively. Fast neutrons markedly induced apoptotic changes in immature hippocampal cells with increased expression of active caspase-3 and cleaved poly(ADP-ribose) polymerase. Increased cytotoxicity and decreased cell viability in immature hippocampal cells were seen in a dose-dependent pattern after fast-neutron and ? irradiation. Fast neutrons have a higher RBE for cell death indices than ? rays. PMID:21692651

Yang, M; Kim, J S; Son, Y; Kim, J; Kim, J Y; Kim, S H; Kim, J C; Shin, T; Moon, C

2011-09-01

376

3D cell culture systems: advantages and applications.  

PubMed

Cell cultures are important material of study for the variety of advantages that they offer. Both established continuous cell lines and primary cell cultures continue to be invaluable for basic research and for direct applications. Technological advancements are necessary to address emerging complex challenges and the way cells are cultured in vitro is an area of intense activity. One important advancement in cell culture techniques has been the introduction of three dimensional culture systems. This area is one of the fastest growing experimental approaches in life sciences. Augmented with advancements in cell imaging and analytical systems, as well as the applications of new scaffolds and matrices, cells have been increasingly grown as three dimensional models. Such cultures have proven to be closer to in vivo natural systems, thus proving to be useful material for many applications. Here, we review the three dimensional way of culturing cells, their advantages, the scaffolds and matrices currently available, and the applications of such cultures in major areas of life sciences. PMID:24912145

Ravi, Maddaly; Paramesh, V; Kaviya, S R; Anuradha, E; Solomon, F D Paul

2015-01-01

377

Bovine ephemeral fever virus in cell culture and mice  

Microsoft Academic Search

Summary Light, immunofluorescent and electron microscopic observations were carried out sequentially on mice and VERO cell cultures infected with bovine ephemeral fever (BEF) virus. In early harvests from cell culture, 185×73 nm cone-shaped particles with nearly parallel sides predominated; these particles had all other features typical of the Rhabdoviruses (surface projections, envelope, axial channel, precisely coiled helical nucleocapsid with 35

Frederick A. Murphy; William P. Taylor; Cedric A. Mims; Sylvia G. Whitfield

1972-01-01

378

Continuous cultures of fused cells secreting antibody of predefined specificity  

Microsoft Academic Search

THE manufacture of predefined specific antibodies by means of permanent tissue culture cell lines is of general interest. There are at present a considerable number of permanent cultures of myeloma cells1,2 and screening procedures have been used to reveal antibody activity in some of them. This, however, is not a satisfactory source of monoclonal antibodies of predefined specificity. We describe

G. Köhler; C. Milstein

1975-01-01

379

High-Aspect-Ratio Rotating Cell-Culture Vessel  

NASA Technical Reports Server (NTRS)

Cylindrical rotating cell-culture vessel with thin culture-medium layer of large surface area provides exchange of nutrients and products of metabolism with minimal agitation. Rotation causes averaging of buoyant forces otherwise separating components of different densities. Vessel enables growth of cells in homogeneous distribution with little agitation and little shear stress.

Wolf, David A.; Sams, Clarence; Schwarz, Ray P.

1992-01-01

380

Regulation of heme metabolism in normal and sideroblastic bone marrow cells in culture  

SciTech Connect

Heme metabolism was examined in developing in vitro erythroid colonies (CFUE) and in bone marrow samples taken directly from four normal donors and four patients with sideroblastic anemia. Maximum activities of delta-aminolevulinic acid synthase (ALAS), ALA dehydratase (ALAD), and /sup 14/C-ALA incorporation into heme were achieved in normal marrow CFUE after 8 days of culture, whereas heme oxygenase progressively decreased to low levels of activity during the same period. Assays on nucleated bone marrow cells taken directly from patients revealed that ALAS activity was considerably reduced in idiopathic sideroblastic anemia (IASA) and X-linked sideroblastic anemia (X-SA) bone marrow specimens, whereas the activity increased more than twofold (normal levels) when cells were assayed from 8-day CFUE. In all cases, ALAD activity appeared to be within normal levels. Measurement of heme synthesis revealed that normal levels of /sup 14/C-ALA incorporation into heme were achieved in IASA cells but were reduced in X-SA cells. In marked contrast to levels in normal cells, heme oxygenase was found to be significantly elevated (two- to fourfold) in bone marrow cells taken directly from patients with IASA and X-SA. Results from this study demonstrate that IASA and X-SA bone marrow cells have disturbances in ALAS and heme metabolism, and that erythropoiesis (CFUE) can be restored to normal levels when cells are cultured in methylcellulose.

Ibraham, N.G.; Lutton, J.D.; Hoffman, R.; Levere, R.D.

1985-05-01

381

Immunopanning purification and long-term culture of human retinal ganglion cells  

PubMed Central

Purpose To establish a robust method to isolate primary retinal ganglion cells (RGCs) from human fetal retina for long-term culture while maintaining neuronal morphology and marker protein expression. Methods A total of six human retinas were obtained from aborted fetuses at 10 to 12 weeks of gestation with informed consent from mothers. RGCs were isolated and purified by a modified two-step immunopanning procedure. The cells were maintained in a serum-free defined medium supplemented with brain-derived neurotrophic factor, ciliary neutrophic factor, and forskolin. The viable RGCs and the extent of neurite outgrowth were examined by calcein-acetoxymethylester assay. Expression of RGC markers was studied by immunocytochemistry. Results Primary RGCs from human fetal retinas were isolated and maintained in vitro for one month with substantial neurite elongation. In cell culture, almost 70% of the isolated cells attached, spread, and displayed numerous dendrites. They were immunoreactive to RGC-specific markers (Thy-1, TUJ-1, and Brn3a) and negative for glial fibrillary acidic protein and amacrine cells marker HPC-1. Conclusions Human RGCs were successfully isolated and maintained in long-term culture. This can serve as an ideal model for biologic, toxicological, and genomic assays of human RGCs in vitro. PMID:21203402

Zhang, Xin-Mei; Li Liu, David Ta; Chiang, Sylvia Wai-Yee; Choy, Kwong-Wai; Pang, Chi-Pui; Lam, Dennis Shun-Chiu

2010-01-01

382

Biona-C Cell Culture pH Monitoring System  

NASA Technical Reports Server (NTRS)

Sensors 2000! is developing a system to demonstrate the ability to perform accurate, real-time measurements of pH and CO2 in a cell culture media in Space. The BIONA-C Cell Culture pH Monitoring System consists of S2K! developed ion selective sensors and control electronics integrated with the fluidics of a cell culture system. The integrated system comprises a "rail" in the Cell Culture Module (CCM) of WRAIR (Space Biosciences of Walter Read Army Institute of Research). The CCM is a Space Shuttle mid-deck locker experiment payload. The BIONA-C is displayed along with associated graphics and text explanations. The presentation will stimulate interest in development of sensor technology for real-time cell culture measurements. The transfer of this technology to other applications will also be of interest. Additional information is contained in the original document.

Friedericks, C.

1999-01-01

383

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

PubMed

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. PMID:22933153

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

2012-01-01

384

Improvement of 96-well microplate assay for estimation of cell growth and inhibition of Leishmania with Alamar Blue.  

PubMed

The value of resazurin-based Alamar Blue redox indicator to determine multiplication of Leishmania promastigotes in 96-well microtiter plates was examined. In addition, assay was validated with amphotericin B (AmB) and allicin. The method was tested on L.donovani and L.infantum promastigotes under different culture conditions (variable air-phase, presence of phenol red, initial cell density, incubation time, use of Hepes buffer). Results showed that the gas-phase of promastigote cultures was critical. The method yielded consistent results with initial plating cell densities of 2.5 × 10? promastigotes/well, up to 72 h incubation and 5% CO? atmosphere or reduced air availability (sealed plastic bags, film-sealed microplates). Detection of low numbers of promastigotes and earlier results could be obtained using fluorimetry instead of spectrophotometry. The addition of 20 mM Hepes improved the results. Fluorescence intensity correlated to promastigotes number in both Leishmania spp. Inhibitory concentration (IC??) values for AmB and allicin using cell counting and fluorimetry were comparable. Under these conditions this one-step, low-cost redox indicator can be used in drug sensitivity assays and studies of differential proliferation rates of Leishmania isolates or strains in a 96-well format. PMID:23707202

Corral, María Jesús; González, Elena; Cuquerella, Montserrat; Alunda, José María

2013-08-01

385

Microsystems platforms for array-based single-cell biological assays  

E-print Network

For much of the past century, plated cell cultures have served investigations regarding a variety of fundamental biological processes. Though this in vitro approach has been fruitful, for surveying topics including cell ...

Taff, Brian M., 1978-

2008-01-01

386

Differentiation of islet cells in long-term culture.  

PubMed

Our previous studies in the hamster pancreatic cancer model have shown that exocrine pancreatic cancer arises from ductal/ductular cells, as well as from within the islets, most probably from islet precursor (stem) cells. To identify and characterize these cells, we established a long-term culture from isolated hamster islets and investigated their growth, differentiation, and expression of biomarkers. Islets maintained their original form and structure within the first 14 days in culture. However, beginning at day 7, ductular structures began to form within the islets. At day 21 in culture, acinar cells, intermediary cells, oncocytes, and cells comparable to pancreatic hepatocytes also appeared between ductular and endocrine cells. The number of duct-like cells gradually increased, whereas the number of hormone-producing cells decreased. After 35 days in culture, the exocrine cells disappeared, and undifferentiated cells formed a monolayer. These cells expressed cytokeratins, alpha1-antitrypsin, transforming growth factor-alpha, epidermal growth factor receptor, carbonic anhydrase II, vimentin, laminin, and showed binding to tomato lectin and Phaseolus vulgaris leukoagglutinin. They did not express the regulatory transcriptional factors, insulin-promoting factor 1, NKx6.1, Pax6, and NeuroD. The results thus indicate that islet cells have potential to form exocrine cells. At present, it is not clear whether these cells originate from preexisting stem cells or from transdifferentiated islet cells. PMID:10824687

Schmied, B M; Liu, G; Matsuzaki, H; Ulrich, A; Hernberg, S; Moyer, M P; Weide, L; Murphy, L; Batra, S K; Pour, P M

2000-05-01

387

UDP-glucose:digitoxin 16'-O-glucosyltransferase from suspension-cultured Digitalis lanata cells.  

PubMed

Suspension cultures from several cell lines of Digitalis lanata, as well as cultures from 6 other plant species were checked for their ability to form purpurea-glycoside A from digitoxin. An in-vitro assay for the UDP-glucose:digitoxin 16'-O-glucosyltransferase (DGT, EC 2.4.1.-) has been established based on an HPLC method. The enzyme is located in the soluble fraction. Its pH optimum is at 7.4. No enzyme activity was found in either purified vacuole preparations or lysed vacuoles. Ascorbate (10 mM) increased the transferase activity about 4-fold. Of the sugar nucleotides tested, only UDP-glucose served as a glucosyl donor. Digitoxin, digoxin, ? -acetyldigitoxin, and ?-acetyldigoxin are substrates for the glucosyltransferase. The role of the DGT during the biotransformation of cardenolides in Digitalis lanata cell suspension cultures is discussed. PMID:24248401

Kreis, W; May, U; Reinhard, E

1986-12-01

388

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

SciTech Connect

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

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

1988-01-01

389

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

PubMed

Several flow cytometric methods for the measurement of circulating immune complexes (CIC) have recently become available. We report a Raji cell flow cytometric assay (FCMA) that uses aggregated human globulin (AHG) as primary calibrator. Technical advantages of the Raji cell flow cytometric assay are discussed, and its clinical usefulness is evaluated in a method comparison study with the widely used Raji cell immunoradiometric assay. FCMA is more precise and has greater analytic sensitivity for AHG. Diagnostic sensitivity by the flow cytometric method is superior in systemic lupus erythematosus (SLE), rheumatoid arthritis, and vasculitis patients: however, diagnostic specificity is similar for both assays, but the reference interval of FCMA is narrower. Significant correlations were found between CIC levels obtained with both methods in SLE, rheumatoid arthritis, and vasculitis patients and in longitudinal studies of two patients with cerebral SLE. The Raji cell FCMA is recommended for measurement of CIC levels to clinical laboratories with access to a flow cytometer. PMID:3219778

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

1988-01-01

390

Tension, Free Space, and Cell Damage in a Microfluidic Wound Healing Assay  

E-print Network

We use a novel, microfluidics-based technique to deconstruct the classical wound healing scratch assay, decoupling the contribution of free space and cell damage on the migratory dynamics of an epithelial sheet. This method ...

Murrell, Michael

391

Incidence of Listeria species in bovine, ovine, caprine, camel and water buffalo milk using cultural method and the PCR assay  

PubMed Central

Objective To determine the prevalence rate of Listeria species in bovine, ovine, caprine, camel and water buffalo milk in Iran. Methods From September 2010 to December 2011 a total of 260 bulk milk samples including 85 bovine, 37 camel, 34 water buffalo, 56 ovine and 48 caprine bulk milk samples were collected from commercial dairy herds, in Fars and Khuzestan provinces, Iran and were evaluated for the presence of Listeria species using cultural method and the PCR assay. Results Using cultural method, 19 samples (7.3%) were positive for Listeria spp. The highest prevalence of Listeria was found in raw water buffalo milk (11.8%), followed by raw bovine milk (10.6%), raw ovine milk (7.1%), and raw caprine milk (4.2%) samples. All 37 camel milk samples from 20 camel breeding farms were negative for Listeria spp. The overall prevalence of Listeria was 7.3%, in which Listeria innocua was the most recovered species (4.2%); the remaining isolates were Listeria monocytogenes (1.9%), Listeria ivanovii (0.08%) and Listeria seeligari (0.04%). The PCR assay could identify 8 Listeria-contaminated milk samples that were negative using the cultural method. Conclusions The results presented in this study indicate the potential risk of infection with Listeria in people consuming raw and unpasteurized milk.

Rahimi, Ebrahim; Momtaz, Hassan; Behzadnia, Asma; Baghbadorani, Zeinab Torki

2014-01-01

392

A quantification of human cells using an ERV-3 real time PCR assay  

Microsoft Academic Search

A novel approach to quantifying human cells using a real time PCR assay was developed. The target sequence used in the assay is a 135 bp segment within the unique 1.7 kb Hind III \\/ Pst I fragment of the ERV-3 envelope gene. ERV-3 is a full-length human endogenous retrovirus present in known copy number in all human cells. The

Chiu Chin Yuan; Wendell Miley; David Waters

2001-01-01

393

Comparison of a New Neuraminidase Detection Assay with an Enzyme Immunoassay, Immunofluorescence, and Culture for Rapid Detection of Influenza A and B Viruses in Nasal Wash Specimens  

Microsoft Academic Search

The performance of a new, rapid, easy-to-perform assay based on neuraminidase enzyme activity for detection of influenza virus types A and B was compared to detection by culture, indirect immunofluorescence, and enzyme immunoassay in 479 nasal wash specimens from children with respiratory infections. Compared to isolation of influenza virus by culture, the neuraminidase assay had a sensitivity of 70.1%, specificity

DANIEL E. NOYOLA; BRUCE CLARK; FREDERICK T. O'DONNELL; ROBERT L. ATMAR; JEWEL GREER; GAIL J. DEMMLER

2000-01-01

394

Evaluation of a Triplex PCR Assay To Discriminate Staphylococcus aureus from Coagulase-Negative Staphylococci and Determine Methicillin Resistance from Blood Cultures  

PubMed Central

A triplex PCR targeting the 16S rRNA, mecA, and nuc genes was developed for identification of staphylococci and detection of methicillin resistance. After validation of the assay with a collection of strains of staphylococci and enterococci (n = 169), the assay was evaluated with cultures of blood with gram-positive cocci from 40 patients. Accurate results were obtained for 59 (98%) of 61 cultures within 6 h of growth detection. PMID:11923385

Maes, N.; Magdalena, J.; Rottiers, S.; De Gheldre, Y.; Struelens, M. J.

2002-01-01

395

Microfluidic biomechanical assay for red blood cells parasitized by Plasmodium falciparum  

E-print Network

Microfluidic biomechanical assay for red blood cells parasitized by Plasmodium falciparum Quan Guo December 2011 DOI: 10.1039/c2lc20857a Red blood cells parasitized by Plasmodium falciparum can and simplified analysis, we developed a microfluidic device to measure red blood cell deformability using

Ma, Hongshen

396

MORPHOLOGICAL-BASED ADAPTIVE SEGMENTATION AND QUANTIFICATION OF CELL ASSAYS IN HIGH CONTENT SCREENING  

E-print Network

, image processing software is necessary to have automatic algorithms for segmenting the cells individ, watershed transformation and granulometries for seg- menting cells of different size, contrast, etc. In particular, the performance of the algorithms is illustrated with cell im- ages from a toxicity assay

Angulo,Jesús

397

Convenient cell fusion assay for rapid screening for HIV entry inhibitors  

NASA Astrophysics Data System (ADS)

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.

Jiang, Shibo; Radigan, Lin; Zhang, Li

2000-03-01

398

Slight Changes in the Mechanical Stimulation Affects Osteoblast- and Osteoclast-Like Cells in Co-Culture  

PubMed Central

Summary Background Osteoblast- and osteoclast-like cells are responsible for coordinated bone maintenance, illustrated by a balanced formation and resorption. Both parameters appear to be influenced by mechanical constrains acting on each of these cell types individually. We hypothesized that the interactions between both cell types are also influenced by mechanical stimulation. Methods Co-cultures of osteoblast- and osteoclast-like cells were stimulated with 1,100 µstrain, 0.1 or 0.3 Hz for 1–5 min/day over 5 days. Two different setups depending on the differentiation of the osteoclast-like cells were used: i) differentiation assay for the fusion of pre-osteoclasts to osteoclasts, ii) resorption assay to determine the activity level of osteoclast-like cells. Results In the differentiation assay (co-culture of osteoblasts with unfused osteoclast precursor cells) the mechanical stimulation resulted in a significant decrease of collagen-1 and osteocalcin produced by osteoblast-like cells. Significantly more TRAP-iso5b was measured after stimulation for 3 min with 0.1 Hz, indicating enhanced osteoclastogenesis. In the resorption assay (co-culture of osteoblasts with fused osteoclasts) the stimulation for 3 min with 0.3 Hz significantly increased the resorption activity of osteoclasts measured by the pit formation and the collagen resorption. The same mechanical stimulation resulted in an increased collagen-1 production by the osteoblast-like cells. The ratio of RANKL/OPG was not different between the groups. Conclusion These findings demonstrate that already small changes in duration or frequency of mechanical stimulation had significant consequences for the behavior of osteoblast- and osteoclast-like cells in co-culture, which partially depend on the differentiation status of the osteoclast-like cells. PMID:24474895

Kadow-Romacker, Anke; Duda, Georg N.; Bormann, Nicole; Schmidmaier, Gerhard; Wildemann, Britt

2013-01-01

399

Droplet optofluidic imaging for ?-bacteriophage detection via co-culture with host cell Escherichia coli.  

PubMed

Bacteriophages are considered as attractive indicators for determining drinking water quality since its concentration is strongly correlated with virus concentrations in water samples. Previously, bacteriophage detection was based on a plague assay that required a complicated labelling technique and a time-consuming culture assay. Here, for the first time, a label-free bacteriophage detection is reported by using droplet optofluidic imaging, which uses host-cell-containing microdroplets as reaction carriers for bacteriophage infection due to a higher contact ratio. The optofluidic imaging is based on the effective refractive index changes in the microdroplet correlated with the growth rate of the infected host cells, which is highly sensitive, i.e. can detect one E. coli cell. The droplet optofluidic system is not only used in drinking water quality monitoring, but also has high potential applications for pathogenic bacteria detection in clinical diagnosis and food industry. PMID:25008551

Yu, J Q; Huang, W; Chin, L K; Lei, L; Lin, Z P; Ser, W; Chen, H; Ayi, T C; Yap, P H; Chen, C H; Liu, A Q

2014-09-21

400

Microglial cells in astroglial cultures: a cautionary note  

PubMed Central

Primary rodent astroglial-enriched cultures are the most popular model to study astroglial biology in vitro. From the original methods described in the 1970's a great number of minor modifications have been incorporated into these protocols by different laboratories. These protocols result in cultures in which the astrocyte is the predominant cell type, but astrocytes are never 100% of cells in these preparations. The aim of this review is to bring attention to the presence of microglia in astroglial cultures because, in my opinion, the proportion of and the role that microglial cells play in astroglial cultures are often underestimated. The main problem with ignoring microglia in these cultures is that relatively minor amounts of microglia can be responsible for effects observed on cultures in which the astrocyte is the most abundant cell type. If the relative contributions of astrocytes and microglia are not properly assessed an observed effect can be erroneously attributed to the astrocytes. In order to illustrate this point the case of NO production in activated astroglial-enriched cultures is examined. Lipopolysaccharide (LPS) induces nitric oxide (NO) production in astroglial-enriched cultures and this effect is very often attributed to astrocytes. However, a careful review of the published data suggests that LPS-induced NO production in rodent astroglial-enriched cultures is likely to be mainly microglial in origin. This review considers cell culture protocol factors that can affect the proportion of microglial cells in astroglial cultures, strategies to minimize the proportion of microglia in these cultures, and specific markers that allow the determination of such microglial proportions. PMID:17937799

Saura, Josep

2007-01-01