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1

Biochemical Assays of Cultured Cells  

NASA Technical Reports Server (NTRS)

Subpopulations of human embryonic kidney cells isolated from continuous flow electrophoresis experiments performed at McDonnell Douglas and on STS-8 have been analyzed. These analyses have included plasminogen activator assays involving indirect methodology on fibrin plated and direct methodology using chromogenic substrates. Immunological studies were performed and the conditioned media for erythropoietin activity and human granulocyte colony stimulating (HGCSF) activity was analyzed.

Barlow, G. H.

1985-01-01

2

Cell Culture Assay for Human Noroviruses [response  

SciTech Connect

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

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

2007-07-01

3

Aquatic toxicity evaluated using human and monkey cell culture assays  

SciTech Connect

A new bioassay using cultured mammalian cells was reported. In this report, the author describes the use of a cell culture assay using growing cells as a screen for aquatic toxicity. Cadmium, copper and zinc toxicity were measured.

Mochida, K.

1986-04-01

4

Robotic cell culture system for stem cell assays  

Microsoft Academic Search

Purpose – The purpose of this paper is to focus on the advantages of a robotic time-lapsed microscopic imaging system for tracking stem cells in in vitro biological assays which measure stem cell activities. Design\\/methodology\\/approach – The unique aspects of the system include robotic movement of stem cell culture flasks which enables selection of a large number of regions of

Benjamin T. Schmidt; Joseph M. Feduska; Ashley M. Witt; Bridget M. Deasy

2008-01-01

5

Microcystis aeruginosa toxin: cell culture toxicity, hemolysis, and mutagenicity assays.  

PubMed Central

Crude toxin was prepared by lyophilization and extraction of toxic Microcystis aeruginosa from four natural sources and a unicellular laboratory culture. The responses of cultures of liver (Mahlavu and PCL/PRF/5), lung (MRC-5), cervix (HeLa), ovary (CHO-K1), and kidney (BGM, MA-104, and Vero) cell lines to these preparations did not differ significantly from one another, indicating that toxicity was not specific for liver cells. The results of a trypan blue staining test showed that the toxin disrupted cell membrane permeability within a few minutes. Human, mouse, rat, sheep, and Muscovy duck erythrocytes were also lysed within a few minutes. Hemolysis was temperature dependent, and the reaction seemed to follow first-order kinetics. Escherichia coli, Streptococcus faecalis, and Tetrahymena pyriformis were not significantly affected by the toxin. The toxin yielded negative results in Ames/Salmonella mutagenicity assays. Microtiter cell culture, trypan blue, and hemolysis assays for Microcystis toxin are described. The effect of the toxin on mammalian cell cultures was characterized by extensive disintegration of cells and was distinguishable from the effects of E. coli enterotoxin, toxic chemicals, and pesticides. A possible reason for the acute lethal effect of Microcystis toxin, based on cytolytic activity, is discussed. Images PMID:6808921

Grabow, W O; Du Randt, W C; Prozesky, O W; Scott, W E

1982-01-01

6

Massively parallel reporter assays in cultured mammalian cells.  

PubMed

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish. PMID:25177895

Melnikov, Alexandre; Zhang, Xiaolan; Rogov, Peter; Wang, Li; Mikkelsen, Tarjei S

2014-01-01

7

Cultured stem cells as tools for toxicological assays.  

PubMed

In the last 2 decades, cell culture techniques for both mammalian embryonic stem cells and adult stem cells have developed and improved, and are now widely available. These stem cells are either pluri- or multi-potent, which makes them favorable for use in vitro developmental toxicity assays. Recent studies have reported several applications for embryonic and adult stem cells in cytotoxicity and developmental toxicity testing. These applications have the potential to provide alternative assessment techniques for evaluating toxic substances, and possibly reveal novel toxic and developmental effects that are difficult to investigate in humans because of ethical considerations. In this review, we describe some of the new approaches that use mammalian embryonic and adult stem cells in toxicological safety testing. PMID:23827858

Mori, Hideki; Hara, Masayuki

2013-12-01

8

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

9

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

10

Gonococcal and meningococcal pathogenesis as defined by human cell, cell culture, and organ culture assays.  

PubMed Central

Human cells, cell cultures, and organ cultures have been extremely useful for studying the events that occur when gonococci and meningococci encounter human mucosal surfaces. The specificity and selectivity of these events for human cells are striking and correlate with the adaptation of these pathogens for survival on human mucous membranes. To colonize these sites, meningococci and gonococci have developed mechanisms to damage local host defenses such as the mucociliary blanket, to attach to epithelial cells, and to invade these cells. Attachment to epithelial cells mediated by pili, and to some types of cells mediated by PIIs, serves to anchor the organism close to sources of nutrition and allows multiplication. Intracellular invasion, possibly initiated by the major porin protein, may provide additional nutritional support and protection from host defenses. Mucosal invasion may also result in access of gonococci and meningococci to the bloodstream, leading to dissemination. Images PMID:2497953

Stephens, D S

1989-01-01

11

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

E-print Network

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 ...

Shin, Yoojin

12

Biochemical assays of cultured cells. [space shuttle oft-3  

NASA Technical Reports Server (NTRS)

Assay systems were developed for use in interpreting samples to be returned on the space shuttle OFT-3 flights. Samples from electrophoretic separation were used to evaluate the techniques. All assays were determinable on the growth media. Approaches are described for assaying: (1) the human granulocyte conditioning factor; (2) urokinase activity; (3) erythropoietin; (4) the molecular form of urokinase; and (5) protein distribution. Other studies are planned to validate that the activity observed is urokinase and not that of other activators or proteases.

Barlow, G. H.

1981-01-01

13

In Vitro Cell Culture Infectivity Assay for Human Noroviruses  

Microsoft Academic Search

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

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

2007-01-01

14

Cell culture assays to evaluate bacterial toxicity and virulence.  

PubMed

Helicobacter pylori CagA and VacA are two critical virulence factors that modulate disease severity in the infected host. The following chapter outlines methods employed to study the effects of these virulence factors on several key signaling pathways in epithelial cells. PMID:23015494

Raju, Deepa; Rizzuti, David; Jones, Nicola L

2012-01-01

15

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

16

Discovering and Differentiating New and Emerging Clonal Populations of Chlamydia trachomatis with a Novel Shotgun Cell Culture Harvest Assay  

Microsoft Academic Search

Chlamydia trachomatis is the leading cause of prevent- able blindness and bacterial sexually transmitted diseases worldwide. Plaque assays have been used to clonally segre- gate laboratory-adapted C. trachomatis strains from mixed infections, but no assays have been reported to segregate clones from recent clinical samples. We developed a novel shotgun cell culture harvest assay for this purpose because we found

Naraporn Somboonna; Sally Mead; Jessica Liu; Deborah Dean

2008-01-01

17

Simplification of aggregate culture of human mesenchymal stem cells as a chondrogenic screening assay  

PubMed Central

Aggregate culture provides a three-dimensional (3-D) environment for differentiating or differentiated cells; it is particularly useful to study in vitro chondrogenesis and cartilage biology. We have recently ported this method from a conical tube-based format to a 96-well plate format for the study of mesenchymal stem cell (MSC) chondrogenesis. The microplate format has greatly reduced the workload and materials cost, while maintaining reproducible chondrogenic differentiation. A long-term goal is to fully automate aggregate culture—this requires critically identifying all the indispensable steps of the protocol. Robotic laboratory equipment for manipulating microplate assays are commercially available; however, centrifugation steps are difficult to implement automatically. We, therefore, tested whether the centrifugation step can be eliminated, thus significantly streamlining the assay work-flow. By comparing aggregates prepared from human bone marrow-derived MSCs (hMSCs) that were formed either through centrifugation or through free sedimentation, we found that both methods produce aggregates with similar formation kinetics, and that there was no perceptible difference in the timing of the appearance of markers of chondrogenesis. Thus, it appears safe to eliminate the centrifugation step from the aggregate culture protocol. This results in significant time and effort savings and paves the way for future full automation of the aggregate assay. PMID:17612296

Welter, Jean F.; Solchaga, Luis A.; Penick, Kitsie J.

2011-01-01

18

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

19

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

PubMed

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

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

2002-03-01

20

Viability staining of soybean suspension-cultured cells and a seedling stem cutting assay to evaluate phytotoxicity of Fusarium solani f. sp. glycines culture filtrates  

Microsoft Academic Search

The phytotoxicity of culture filtrates of Fusarium solani f. sp. glycines, the fungus causing sudden death syndrome (SDS) of soybean (Glycine max), was tested with a viability stain of soybean suspension-cultured cells and a stem cutting assay of soybean seedlings. Suspension-cultured\\u000a cells from a SDS-susceptible soybean cultivar were exposed to cell-free culture filtrates of F. solani f. sp. glycines or

S. Li; G. L. Hartman; J. M. Widholm

1999-01-01

21

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

22

Comparison of a Commercial Real-Time PCR Assay for tcdB Detection to a Cell Culture Cytotoxicity Assay and Toxigenic Culture for Direct Detection of Toxin-Producing Clostridium difficile in Clinical Samples  

Microsoft Academic Search

Rapid detection of toxin-producing strains of Clostridium difficile is essential for optimal management of patients with C. difficile infection. The BD GeneOhm (San Diego, CA) Cdiff assay, a real-time PCR assay that amplifies tcdB, was compared to a cell culture neutralization assay (Wampole C. difficile Toxin B (TOX-B) test; TechLab, Blacksburg, VA) and to toxigenic culture. Using liquid (n 273)

Paul D. Stamper; Romina Alcabasa; Deborah Aird; Wisal Babiker; Jennifer Wehrlin; Ijeoma Ikpeama; Karen C. Carroll

23

DNA damage and repair measurements from cryopreserved lymphocytes without cell culture--a reproducible assay for intervention studies.  

PubMed

Single-cell gel electrophoresis (the Comet assay) can be used to measure DNA damage and DNA repair capacity (DRC). However, to test DRC of cryopreserved lymphocytes, published methods include steps for cell culturing and phytohemagglutinin stimulation, which may limit use of this assay in intervention studies. We developed a modified Comet assay protocol that allows us to measure DRC from cryopreserved lymphocytes without these in vitro manipulations. Assay reproducibility was evaluated by performing the assay six times on different dates using six aliquots from one blood draw of one individual. The interindividual variation was assessed by performing the assay using one aliquot from six individuals. When gamma-irradiation was used as the mutagen, intra-assay coefficients of variation (CVs.) for baseline DNA damage, damage after gamma-irradiation exposure, and DRC--measured as tail moment--were 8, 31, and 10%, respectively. Interindividual CVs. were higher. When H(2)O(2) was used as the mutagen, intra-assay CVs. for damage measurements were lower for a protocol modification that included damage and repair at 37 degrees C (CVs. ranging from 8 to 35%) than for the more standard 4 degrees C protocol. Analyzing moment arm--the average distance of DNA migration within the tail--yielded similar results. DNA repair was successfully detected in each experiment. Comparing freshly isolated lymphocytes to cryopreserved lymphocytes from the same individuals' blood draw indicated that DRC was highly correlated when determined using moment arm values. This modified protocol extends the use of the Comet assay to measuring DRC in intervention studies (e.g., dietary interventions) in that it assesses cellular response after cryopreservation without cell culture or other extensive manipulation. PMID:16673412

Chang, Jyh-Lurn; Chen, Gang; Lampe, Johanna W; Ulrich, Cornelia M

2006-08-01

24

Improved chicken embryo cell culture plaque assay for scrub typhus rickettsiae.  

PubMed Central

The plaque technique for three strains of Rickettsia tsutsugamushi in chicken embryo cell cultures was greatly improved by modifying the trypsinizing procedure and employing homologous chicken serum in the overlay medium. Images PMID:412863

Woodman, D R; Grays, R; Weiss, E

1977-01-01

25

Culturing of cerebellar granule cells to study neuronal migration: gradient and local perfusion assays.  

PubMed

Cultures of cerebellar granule cells are a suitable model to analyze the mechanisms governing neuronal migration. In this unit, we describe a protocol to obtain cultures of dissociated granule cells at a low density, where individual cells can be easily observed. In addition, we include a protocol for studying neuronal migration in these cultures, using single, actively migrating cerebellar granule cells. Following this protocol, a factor of interest can be applied either in a gradient concentration by means of a micropipet located near the neuron, or in a homogeneous concentration by locally perfusing a certain region of the neuron. Time-lapse images are taken to analyze changes in the speed and/or directionality of the observed neuron. Overall, the two protocols take more or less a day and a half to perform, and are a useful way to evaluate a certain factor/drug for its chemotactic activity or its capacity to alter migration speed. PMID:22752893

Guijarro, Patricia; Jiang, Jian; Yuan, Xiao-bing

2012-07-01

26

Detoxification of exogenous hydrogen peroxide and organic hydroperoxides by cultured astroglial cells assessed by microtiter plate assay.  

PubMed

Peroxides are often applied to cultured brain cells to investigate functions of these cells under oxidative stress. However, little is known about the ability of brain cells to detoxify peroxides. In order to investigate peroxide clearance of adherent cultured cells, the peroxide assay originally described for the determination of hydrogen peroxide production during experimental protein glycation by Jiang et al. [Z.-Y. Jiang, A.C.S. Woollard, S.P Wolff, Hydrogen peroxide production during experimental protein glycation, FEBS Lett. , 268 (1990) 69-71.] was adapted to microtiter plates. Besides hydrogen peroxide, with this assay organic hydroperoxides such as tertiary butylhydroperoxide (tBHP), and cumene hydroperoxide (CHP) can also be quantified. Up to an amount of 2.5 nmol of each peroxide per well of a plate the absorption measured was proportional to the concentration of the peroxide. Using the assay described the ability of astroglia-rich primary cultures to detoxify peroxides was monitored by measuring the peroxide content in 10 microliter samples collected at several time points from the peroxide-containing incubation buffer of one dish. If peroxides were applied at a concentration of 100 muM, hydrogen peroxide, tBHP, and CHP disappeared from the incubation buffer in reactions following first order kinetics with apparent half-times of 3.1 min, 2.9 min, and 4.2 min, respectively. In the absence of cells H2O2 and CHP were stable in the incubation buffer for at least 30 min, whereas tBHP decayed slowly in a spontaneous reaction. In conclusion, the method presented allows the determination of the rapid detoxification of various peroxides by cultured cells. PMID:9507143

Dringen, R; Kussmaul, L; Hamprecht, B

1998-03-01

27

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

28

Hormone dependency of human breast neoplasms cultured in vitro in the stem cell assay.  

PubMed

The stem cell soft agar culture system provides a biological tool with which to study tumor clones derived from heterogeneous tumor cell populations. For testing the feasibility of this approach in identifying hormone-responsive clones of mammary tumors, a study was done to determine whether estrogen deprivation or supplementation would alter colony formation in sequential breast neoplasms received in the Cell Kinetics Laboratory, The Pennsylvania State University. Single-cell suspensions obtained from 22 freshly excised breast neoplasms (20 malignant, 2 benign) were plated in soft agar with and without tamoxifen (Tam) (10(-7) M) with the use of serum-supplemented media and with and without 17 beta-estradiol (E2) (10(-8) M] under serum-free medium conditions. Twenty tumors successfully grew, producing 42 +/- 8 (mean +/- SEM) colonies in control dishes in the presence of serum and 27 +/- 5 in its absence (P less than .001). Tam inhibited colony formation in 78% of malignant tumors, whereas a stimulatory effect was observed with E2 in 94%. An increase in colony formation was induced by Tam in 4 tumors (2 benign and 2 malignant). The effects of E2 and Tam on tumor growth were not influenced by the estrogen and progesterone receptor status of the tumor. These preliminary results suggest that all tumors, irrespective of their receptor status, may contain clones of hormone-responsive cells. PMID:2985856

Manni, A; Wright, C; Pontari, M; Feil, P; Joehl, R

1985-04-01

29

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

PubMed Central

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

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

2012-01-01

30

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

31

A rapid and specific microplate assay for the determination of intra- and extracellular ascorbate in cultured cells.  

PubMed

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within the brain, ascorbate acts in a neuroprotective and neuromodulatory manner that involves ascorbate cycling between neurons and vicinal astrocytes--a relationship that appears to be crucial for brain ascorbate homeostasis. Additionally, emerging evidence strongly suggests that ascorbate has a greatly expanded role in regulating cellular and systemic iron metabolism than is classically recognized. The increasing recognition of the integral role of ascorbate in normal and deregulated cellular and organismal physiology demands a range of medium-throughput and high-sensitivity analytic techniques that can be executed without the need for highly expensive specialist equipment. Here we provide explicit instructions for a medium-throughput, specific and relatively inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture. PMID:24747535

Lane, Darius J R; Lawen, Alfons

2014-01-01

32

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

PubMed Central

Evaluation of drug cardiotoxicity is essential to the safe development of novel pharmaceuticals. Assessing a compound's risk for prolongation of the surface electrocardiographic QT interval, and hence risk for life threatening arrhythmias is mandated before approval of nearly all new pharmaceuticals. QT prolongation has most commonly been associated with loss of current through hERG (human ether-a-go-go related gene) potassium ion channels due to direct block of the ion channel by drugs or occasionally by inhibition of the plasma membrane expression of the channel protein. To develop an efficient, reliable, and cost-effective hERG screening assay for detecting drug-mediated disruption of hERG membrane trafficking, we demonstrate the use of microfluidic-based systems to improve throughput and lower cost of current methods. We validate our microfluidics array platform in polystyrene (PS), cyclo-olefin polymer (COP) and poly(dimethylsiloxane) (PDMS) microchannels for drug-induced disruption of hERG trafficking by culturing stably transfected HEK cells that overexpressed hERG (WT-hERG), and studying their morphology, proliferation rates, hERG protein expression, and response to drug treatment. Our results show that WT-hERG cells readily proliferate in PS, COP, and PDMS microfluidic channels. We demonstrated that conventional Western blot analysis was possible using cell lysate extracted from a single microchannel. The Western blot analysis also provided important evidence that WT-hERG cells cultured in microchannels maintained regular (well plate-based) expression of hERG. We further showed that experimental procedures can be streamlined by using direct in-channel immunofluorescent staining in conjunction with detection using an infrared scanner. Finally, treatment of WT-hERG cells with five different drugs suggested that PS (and COP) microchannels were more suitable than PDMS microchannels for drug screening applications, particularly for tests involving hydrophobic drug molecules. PMID:21131594

Su, Xiaojing; Young, Edmond W.K.; Underkofler, Heather A. S.; Kamp, Timothy J.; January, Craig T.; Beebe, David J.

2011-01-01

33

Validation of a qPCR assay for the detection of Ictalurid herpesvirus-2 (IcHV-2) in fish tissues and cell culture supernatants.  

PubMed

Ictalurid herpesvirus-2 (IcHV-2) is a pathogen of cultured black bullhead, Ameiurus melas (Rafinesque), and has been shown to produce high mortality in experimental exposures of channel catfish, Ictalurus punctatus (Rafinesque). During acute infections, the virus grows readily in cell cultures but produces a cytopathic effect (CPE) similar to that of Ictalurid herpesvirus-1 (IcHV-1) and the channel catfish reovirus. We have developed a quantitative PCR assay that can be used to detect IcHV-2 in fish tissues and cell culture supernatants. The assay does not amplify other fish herpesviruses tested or host DNA. It is quantitative over a range of eight logs, and the limit of detection is <10 copies per reaction. In replicate assays carried out on different days, the coefficient of variability was 10%. The best organs for the detection of acute IcHV-2 infections by our assay are the spleen and kidney. This assay should be useful for the diagnosis of IcHV-2 disease, the identification of syncytial CPEs in cell cultures, and for the detection of latent infections in carrier fish. PMID:20082661

Goodwin, A E; Marecaux, E

2010-04-01

34

Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity  

Microsoft Academic Search

Retroviral vectors with long terminal re- peats (LTRs), which contain strong en- hancer\\/promoter sequences at both ends of their genome, are widely used for stable gene transfer into hematopoietic cells. However, recent clinical data and mouse models point to insertional activation of cellular proto-oncogenes as a dose- limiting side effect of retroviral gene deliv- ery that potentially induces leukemia. Self-

Ute Modlich; Jens Bohne; Manfred Schmidt; Christof von Kalle; Sabine Knoss; Axel Schambach; Christopher Baum

2006-01-01

35

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

36

Evaluation of potential organ culture media for eye banking using a human corneal endothelial cell growth assay  

Microsoft Academic Search

Background: To evaluate the ability of different commercially available cell culture media to induce proliferation and morphological changes in primary cultures of human corneal endothelial cells (HCEC). This screening model was used in an attempt to establish a rational basis for the development of well-defined, serum-free preservation media for long-term organ culture of human donor corneas. Methods: A total of

Torben Møller-Pedersen; Ulrike Hartmann; Niels Ehlers; Katrin Engelmann

2001-01-01

37

A southern blot assay for detection of hepatitis B virus covalently closed circular DNA from cell cultures.  

PubMed

Chronic hepatitis B remains a substantial public health burden affecting approximately 350 million people worldwide, causing cirrhosis and liver cancer, and about 1 million people die each year from hepatitis B and its complications. Hepatitis B is caused by hepatitis B virus (HBV) infection. As an essential component of the viral life cycle, HBV covalently closed circular DNA (cccDNA) is synthesized and maintained at low copy numbers in the nucleus of infected hepatocytes, and serves as the transcription template for all viral RNAs. Therefore, cccDNA is responsible for the establishment of viral infection and persistence. The presence and longevity of cccDNA may also explain the limitations of current antiviral therapy for hepatitis B. Thus, understanding the mechanisms underlying cccDNA formation and regulation is critical in understanding the HBV pathogenesis and finding a cure for hepatitis B. Here we describe a protocol for HBV cccDNA extraction and detection in detail. The procedure includes two major steps: (1) HBV cccDNA extraction by Hirt protein-free DNA extraction method and (2) HBV cccDNA detection by Southern blot analysis. The method is straightforward and reliable for cccDNA assay with cell culture samples, and it is useful for both HBV molecular biology and antiviral research. PMID:23821267

Cai, Dawei; Nie, Hui; Yan, Ran; Guo, Ju-Tao; Block, Timothy M; Guo, Haitao

2013-01-01

38

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

PubMed Central

An indirect immunofluorescence assay for the detection of human antibodies to the agent of human granulocytic ehrlichiosis (HGE) was developed and standardized. Antigen was prepared from a human promyelocytic leukemia cell line (HL-60) infected with a tick-derived isolate of the HGE agent (USG3). Suitable antigen presentation and preservation of cellular morphology were obtained when infected cells were applied and cultured on the slide, excess medium was removed, and cells were fixed with acetone. Use of a buffer containing bovine serum albumin and goat serum reduced background fluorescence, and use of an immunoglobulin G (gamma-specific) conjugate reduced nonspecific binding. The assay readily detected specific antibody from HGE patients and did not detect antibody from healthy individuals. No significant reactivity was noted in sera from patients with high titers of antibodies to other rickettsial species. We were able to identify antibodies reactive to USG3 antigen in samples from areas where HGE is endemic that had tested negative to other rickettsial agents. Animal sera reactive against Ehrlichia equi or Ehrlichia phagocytophila bound to the HGE antigen, indicating that the assay may be useful for veterinary use. Comparability between two different laboratories was assessed by using coded human sera exchanged between laboratories. Results from the two laboratories were similar, indicating that the assay can be easily integrated into use for routine testing for HGE. The assay was then compared to an assay using horse neutrophils infected with ehrlichiae. The two assays gave comparable results, indicating that the cell culture-derived antigen can be used for testing samples that have been previously tested with E. equi as an antigen. The new assay offers several advantages over other immunofluorescence methods that use animal-derived antigen and is suitable for use in testing for human antibodies to the HGE agent. PMID:9163471

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

1997-01-01

39

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

2012-03-12

40

Analysis of cytotoxicity of melittin on adherent culture of human endothelial cells reveals advantage of fluorescence microscopy over flow cytometry and haemocytometer assay.  

PubMed

Melittin, from the honeybee venom, is a membrane active protein, whose cytotoxicity to human endothelial cells has not been described yet. In this work, we studied its time-dependent cytotoxicity on human umbilical vein endothelial cells (HUVECs). Since HUVECs grow in culture as adherent cells, suspension of cells is required before measuring cytotoxicity with a haemocytometer or flow cytometry. Therefore, we also tried to discover whether the result of cytotoxicity tests of melittin is influenced by the preparation of the cell suspension. For this purpose, we compared the results of haemocytometer-based trypan blue assay and flow cytometry using 7-aminoactinomycin D (7-AAD) with results of fluorescence microscopy using 7-AAD and 4', 6-diamidino-2-phenylindole (DAPI). Melittin over 60 min exposure evoked a rapid decline in the survival of HUVEC. After 60 min exposure to melittin, the phase contrast microscopy demonstrated massive necrosis in the remaining attached cells. Fluorescence microscopy detected both viable and non-viable cells in adequate proportions at all exposure times, whereas haemocytometer-based assay and flow cytometry highly underestimated the percentage of non-viable cells or even failed to detect any dead cells. Our data clearly indicate that the induction of large-scale damage to adherent endothelial cells by melittin results in a loss of the majority of necrotic cells during sample preparation for flow cytometry or a haemocytometer-based assay. In the case of adherent cell culture, therefore, fluorescence microscopy was shown to be a more appropriate method for quantitative analysis of cell death caused by a fast-acting cytolytic toxin such as melittin. PMID:23456458

?erne, Katarina; Erman, Andreja; Verani?, Peter

2013-10-01

41

Gene expression, glycocalyx assay, and surface properties of human endothelial cells cultured on hydrogel matrix with sulfonic moiety: Effect of elasticity of hydrogel.  

PubMed

We measured the gene expression, glycocalyx content, and surface properties of human coronary artery endothelial cells (HCAECs) cultured on poly(sodium p-styrene sulfonate) (PNaSS) hydrogels with various levels of elasticity ranged in 3-300 kPa. We found that all HCAECs reached confluence on these hydrogels while retaining the similar expression of EC-specific markers to that on polystyrene (PS), a widely used scaffold in cell culture in vitro. Real-time polymerase chain reaction (PCR) and glycosaminoglycan (GAG) assay showed that the amount of EC-specific glycocalyx secreted by HCAECs cultured on PNaSS gels was higher than that cultured on PS, and it increased with an increase of gel elasticity. Furthermore, the HCAECs cultured on PNaSS gels showed excellent property against platelet adhesion and lower surface friction than that on PS. The platelet adhesion and surface friction of HCAECs cultured on PNaSS gels also depend on the elasticity of gels. The largest amount of EC-specific glycocalyx, excellent blood compatibility, and the lowest friction were observed when the elastic modulus of the gel was larger than 60 kPa. Overall, HCAECs cultured on these hydrogels have better properties than those cultured on PS scaffold, demonstrating the PNaSS gels can be used as potential tissue engineering material for blood vessels. PMID:20681030

Yang, Jing Jing; Chen, Yong Mei; Kurokawa, Takayuki; Gong, Jian Ping; Onodera, Shin; Yasuda, Kazunori

2010-11-01

42

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

E-print Network

through hERG (human ether-a-go-go related gene) potassium ion channels due to direct block of the ion-mediated disruption of hERG membrane trafficking, we demonstrate the use of microfluidic-based systems to improve of hERG trafficking by culturing stably transfected HEK cells that overexpressed hERG (WT

Beebe, David J.

43

Cell isolation and culture.  

PubMed

Cell isolation and culture are essential tools for the study of cell function. Isolated cells grown under controlled conditions can be manipulated and imaged at a level of resolution that is not possible in whole animals or even tissue explants. Recent advances have allowed for large-scale isolation and culture of primary C. elegans cells from both embryos and all four larval stages. Isolated cells can be used for single-cell profiling, electrophysiology, and high-resolution microscopy to assay cell autonomous development and behavior. This chapter describes protocols for the isolation and culture of C. elegans embryonic and larval stage cells. Our protocols describe isolation of embryonic and L1 stage cells from nematodes grown on high-density NA22 bacterial plates and isolation of L2 through L4 stage cells from nematodes grown in axenic liquid culture. Both embryonic and larval cells can be isolated from nematode populations within 3 hours and can be cultured for several days. A primer on sterile cell culture techniques is given in the appendices. PMID:23430760

Zhang, Sihui; Kuhn, Jeffrey R

2013-01-01

44

Enumerating stem cell frequency: neural colony forming cell assay.  

PubMed

Recent reports have highlighted several parameters of the neurosphere culture or assay system which render it unreliable as a quantitative in vitro assay for measuring neural stem cell (NSC) frequency. The single-step semi-solid based assay, the Neural Colony Forming Cell (NCFC) assay is an assay which was developed to overcome some of the limitations of the neurospheres assay in terms of accurately measuring NSC numbers. The NCFC assay allows the discrimination between NSCs and progenitors by the size of colonies they produce (i.e. their proliferative potential). The NCFC assay and other improved tissue culture tools offer further advances in the promising application of NSCs for therapeutic use. PMID:23934839

Louis, Sharon A; Mak, Carmen K H

2013-01-01

45

Fluorimetric DNA assay of cell number.  

PubMed

This fluorimetric assay has utility for the accurate assessment of cultured epidermal cell numbers by virtue of their deoxyribonucleic acid content, which is the most significant correlate available. The improvement in fluorochromes in the recent past makes PicoGreen the dye of choice for this, with its greatly increased sensitivity (+/- 50 cells) over the Hoechst and DAPI stains and which remains linear over several orders of magnitude with a single dye concentration. The assay involves minimal liquid handling to achieve cell disruption by sodium dodecyl sulfate in saline sodium citrate buffer, and PicoGreen staining is rapidly assayed by a multiwell plate reading fluorimeter, which can be automated for robotic high throughput use. Highly fibrous cells like epidermal keratinocytes can be disrupted using 8 M urea and assayed after dilution. The assay is also compatible with tritiated thymidine incorporation. PMID:15502190

Otto, William R

2005-01-01

46

Advances in cell culture  

SciTech Connect

This book presents papers on advances in cell culture. Topics covered include: Genetic changes in the influenza viruses during growth in cultured cells; The biochemistry and genetics of mosquito cells in culture; and Tree tissue culture applications.

Maramorosch, K. (Dept. of Entomology, Rutgers Univ., New Brunswick, NJ (US))

1987-01-01

47

Kit-On-A-Lid-Assays for accessible self-contained cell assays.  

PubMed

Microscale methods for cell-based assays typically rely on macroscopic reagent handling and fluidic loading protocols that are technically challenging and do not scale with the number of assays favorably. Here, we demonstrate a microfluidic platform technology called "Kit-On-A-Lid-Assay" (KOALA), that enables the creation of self-contained microfluidic cell-based assays, integrating all the steps required to perform cell-based assays. The KOALA platform allows the pre-packaging of reagents, cryopreservation of cell suspensions, thawing of cell suspensions, culture of cells, and operation of whole cell-based assays. The operation of the KOALA platform is user-friendly and consists of bringing together a lid containing the microchannels, and a base containing the pre-packaged reagents, thereby causing fluidic exchange in all the channels simultaneously. We demonstrate that the KOALA cell-based assays can be simply operated from start to finish without any external laboratory equipment. PMID:23229806

Berthier, Erwin; Guckenberger, David J; Cavnar, Peter; Huttenlocher, Anna; Keller, Nancy P; Beebe, David J

2013-02-01

48

Improved Risk Analysis by Dual Direct Detection of Total and Infectious Cryptosporidium Oocysts on Cell Culture in Combination with Immunofluorescence Assay?  

PubMed Central

The inactivation of Cryptosporidium oocysts is a main driver in the selection of water treatment disinfection strategies, and microbial risk analysis provides a sound basis for optimizing water treatment processes. U.S. Environmental Protection Agency method 1622/23 provides an estimate of the total oocyst count; however, it cannot be used directly for risk assessment, as it does not determine the fraction of infectious oocysts. Improved assessment of the risk for designated sources or in treated water requires evaluation of the total number of oocysts and an estimate of their infectivity. We developed a dual direct detection method using differential immunofluorescent staining that allows detection of both oocysts and cell culture infection foci for each sample. Using Cryptosporidium parvum oocysts, various pH levels, proteases, and gastroenteric compounds and substrates were assessed to determine their abilities to enhance the number of infection foci. The results showed that the key trigger for oocyst stimulation was acidification. Addition of a low concentration of d-glucose (50 mM) to the infection media increased rates of infectivity, while a higher dose (300 mM) was inhibitory. The total number of oocysts in each sample was determined by counting the oocysts remaining on a cell monolayer and the oocysts recovered from cell monolayer washes during processing using a simple filtration technique. With the dual direct detection on cell culture with immunofluorescence assay method, it is now possible to determine the numbers of total and infectious oocysts for a given sample in a single analysis. Direct percentages of infectivity are then calculated, which allows more accurate assessments of risk. PMID:19933339

Lalancette, Cindy; Di Giovanni, George D.; Prevost, Michele

2010-01-01

49

Clonogenic assay of endothelial progenitor cells.  

PubMed

In stem cell biology, CD34+ or CD133+ hematopoietic stem cells (HSCs) give rise to two types of endothelial progenitor cell (EPC) colonies: primitive and definitive EPC-colony forming units (primitive EPC-CFU and definitive EPC-CFU), which can be morphologically defined. Based on their morphology, an evaluation of the number or the ratio of each EPC colony constitutes the Endothelial Progenitor Cell Clonogenic Forming Assay (EPC-CFA), a novel assay to quantify the differentiation of colony forming EPCs. This assay system allows us to practically evaluate the vasculogenic potential of primary or cultured stem cell populations, i.e., mononuclear cells or fractionated stem cells (CD34+ or CD133+ cells) in peripheral blood, bone marrow, or umbilical cord blood. EPC-CFA can be used not only for basic research in vascular biology but also for evaluating the vascular reparative activity of patients with cardiovascular diseases. This review summarizes the underlying concepts and significance of the EPC-CFA in vascular biology. PMID:23375595

Masuda, Haruchika; Asahara, Takayuki

2013-05-01

50

Mutation assays involving blood cells that metabolize toxic substances  

SciTech Connect

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. 2 figs.

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

1985-07-30

51

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

52

A biochip platform for cell transfection assays.  

PubMed

In this paper, we describe the development and characterization of a biochip platform for cell transfection assays. Silicon wafers were surface modified by plasma polymerization of allylamine plasma polymer (ALAPP) and grafting of a protein-resistant layer of poly(ethylene oxide) (PEO) on the plasma polymer surface. Excimer laser ablation was then used to pattern ALAPP-PEO coated samples for spatially controlled protein adsorption and subsequent cell attachment. X-ray photoelectron spectroscopy (XPS) was used to characterize the surface modifications before and after excimer laser ablation. Experiments confirmed the creation of a two-dimensionally controlled surface chemistry on the biochip. Cell culture experiments using human embryonic kidney (HEK 293) cells showed that cells attached exclusively to laser ablated areas. In addition, cells confined to ablated areas were successfully transfected with plasmid DNA containing the gene for green fluorescent protein (GFP). The cell transfection efficiencies of cells growing in a culture flask and cells confined on the biochip were determined to be 21 and 13%, respectively. PMID:15093210

Szili, Endre; Thissen, Helmut; Hayes, Jason P; Voelcker, Nicolas

2004-06-15

53

Pumps for microfluidic cell culture.  

PubMed

In comparison to traditional in vitro cell culture in Petri dishes or well plates, cell culture in microfluidic-based devices enables better control over chemical and physical environments, higher levels of experimental automation, and a reduction in experimental materials. Over the past decade, the advantages associated with cell culturing in microfluidic-based platforms have garnered significant interest and have led to a plethora of studies for high throughput cell assays, organs-on-a-chip applications, temporal signaling studies, and cell sorting. A clear concern for performing cell culture in microfluidic-based devices is deciding on a technique to deliver and pump media to cells that are encased in a microfluidic device. In this review, we summarize recent advances in pumping techniques for microfluidic cell culture and discuss their advantages and possible drawbacks. The ultimate goal of our review is to distill the large body of information available related to pumps for microfluidic cell culture in an effort to assist current and potential users of microfluidic-based devices for advanced in vitro cellular studies. PMID:23893649

Byun, Chang Kyu; Abi-Samra, Kameel; Cho, Yoon-Kyoung; Takayama, Shuichi

2014-02-01

54

Stem cell culture engineering  

Microsoft Academic Search

Stem cells have the capacity for self renewal and undergo multilineage differentiation. Stem cells isolated from both blastocysts and adult tissues represent valuable sources of cells for applications in cell therapy, drug screening and tissue engineering. While expanding stem cells in culture, it is critical to maintain their self?renewal and differentiation capacity. In generating particular cell types for specific applications,

Gargi Seth; Catherine M. Verfaillie

2005-01-01

55

Cell Culture Made Easy.  

ERIC Educational Resources Information Center

Outlines steps to generate cell samples for observation and experimentation. The procedures (which use ordinary laboratory equipment) will establish a short-term primary culture of normal mammalian cells. Information on culture vessels and cell division and a list of questions to generate student interest and involvement in the topics are…

Dye, Frank J.

1985-01-01

56

Effects of defined medium, fetal bovine serum, and human serum on growth and chemosensitivities of human breast cancer cells in primary culture: Inference for in vitro assays  

Microsoft Academic Search

Summary  We compared the effects of defined medium, fetal bovine serum (FBS) and human serum (HuS) on the growth and responses to chemotherapeutic\\u000a agents of human breast cancer cells in primary culture. Normal and tumor tissues were dissociated to small aggregates and\\u000a single cells and seeded onto collagen-gel-coated wells in defined medium or medium supplemented with 5% FBS or 5% HuS.

Joanne T. Emerman; Eveline E. Fiedler; Anthony W. Tolcher; Patricia M. Rebbeck

1987-01-01

57

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

58

Mutation assays involving blood cells that metabolize toxic substances  

SciTech Connect

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

59

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.

2014-08-21

60

Fidelity of micropatterned cell cultures.  

PubMed

Methods that enable the culture of micropatterned cells may help advance our fundamental understanding of cell-cell and cell-surface interactions, while facilitating the development and implementation of cell-based biological assays. However, the long-term stability of the cell patterns can limit the time scales over which such methods can be informative. Here we used self-assembling monolayers (SAMs) to localize the adsorption of baby hamster kidney (BHK-21) cells as well as cells from a murine astrocytoma-derived cell line (delayed brain tumor) in linear arrays. We tested the effects of surface chemistries, fibronectin pre-treatments, array dimensions, and cell types on pattern fidelity. Changes in patterns were monitored by phase-contrast microscopy up to 96 h post-plating, followed by digital imaging, and these changes were quantified by measuring an "intrusion distance" or the average distance cells extend beyond the initial adhesive/non-adhesive boundary. Loss of pattern boundaries involved different mechanisms for different cells. Treatment of patterned surfaces with fibronectin prior to plating of cells tended to promote earlier loss of pattern fidelity, and the extent of pattern loss was further augmented for SAMs formed using hydrophobic monolayers. Finally, reduction of gap spacing between adjacent cell arrays promoted pattern loss. PMID:15920741

Endler, Elizabeth E; Nealey, Paul F; Yin, John

2005-07-01

61

Rapidly derived colorectal cancer cultures recapitulate parental cancer characteristics and enable personalized therapeutic assays.  

PubMed

We have developed a simple procedure for deriving pure cultures of growing cancer cells from colorectal cancers, including material refrigerated overnight, for pathological characterization and cytotoxicity assays. Forty-six cancers were processed and cultures set up under varying culture conditions. Use of a Rho kinase (ROCK1) inhibitor markedly increased culture survival, resulting in 80% of samples growing in culture for at least 1 month and beyond. Overnight refrigeration of samples before culture initiation had little effect on success rates, paving the way for cultures to be established for samples collected over wide geographical areas, such as those for clinical trials. Primary cultures demonstrated good correlation for differentiation markers compared to parent cancers, and were highly dynamic in 3D culture. In Matrigel, many colonies formed central lumens, indicating the presence of stem-like cells. Viable colonies in these cultures recapitulated the in vivo generation of carcinoembryonic antigen (CEA)-positive necrotic/apoptotic debris, much of which was derived from abnormal vacuolated dynamic 'bubble cells' that have not previously been described. Although bubble cells morphologically resembled signet ring cells, a rare cancer subtype, immunostaining suggested that they were most likely derived from terminally differentiated enterocytes. Micro-assays showed that drug toxicity could be measured in these cultures within hours and with sensitivity down to a few hundred cells. Primary cultures derived by our method provide valid in vitro avatars for studying the pathology of cancers in vitro and are amenable to pre-clinical drug testing, paving the way for personalized cancer treatment. PMID:24797403

Ashley, Neil; Jones, Matthew; Ouaret, Djamila; Wilding, Jenny; Bodmer, Walter F

2014-09-01

62

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

63

21 CFR 864.7100 - Red blood cell enzyme assay.  

Code of Federal Regulations, 2013 CFR

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

2013-04-01

64

Development and validation of a sensitive assay for the quantification of imatinib using LC/LC-MS/MS in human whole blood and cell culture  

PubMed Central

We developed and validated a semi-automated LC/LC-MS/MS assay for the quantification of imatinib in human whole blood and leukemia cells. After protein precipitation, samples were injected into the HPLC system and trapped onto the enrichment column (flow 5 mL/min); extracts were back-flushed onto the analytical column. Ion transitions [M + H]+ of imatinib (m/z = 494.3 ? 394.3) and its internal standard trazodone (372.5 ? 176.3) were monitored. The range of reliable response was 0.03–75 ng/mL. The inter-day precisions were: 8.4% (0.03 ng/mL), 7.2% (0.1 ng/mL), 6.5% (1 ng/mL), 8.2% (10 ng/mL) and 4.3% (75 ng/mL) with no interference from ion suppression. Autosampler stability was 24 hs and samples were stable over three freeze–thaw cycles. This semi-automated method is simple with only one manual step, uses a commercially available internal standard, and has proven to be robust in larger studies. PMID:19517424

Klawitter, Jelena; Zhang, Yan Ling; Klawitter, Jost; Anderson, Nora; Serkova, Natalie J.; Christians, Uwe

2011-01-01

65

Cell Culturing of Cytoskeleton  

NASA Technical Reports Server (NTRS)

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

2004-01-01

66

Cell Culturing of Cytoskeleton  

NASA Technical Reports Server (NTRS)

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

2004-01-01

67

Novel cultured porcine corneal irritancy assay with reversibility endpoint.  

PubMed

Several alternative assays exist to assess ocular irritancy without the use of live animals. However, these assays cannot address ocular injury reversibility. Reversibility is an issue critical to regulatory authorities and manufactures of commercial products, as ocular irritation caused by misuse or accidental exposure to a product may cause irreversible eye damage. Here we report the development and initial characterization of a novel ocular irritation assay that addresses ocular injury reversibility. This assay, the Porcine Corneal Ocular Reversibility Assay (PorCORA), uses an air-interface porcine corneal culture system to sustain ex vivo porcine corneas as a model system. These corneas are maintained in culture for 21 days to determine if cornea injury, once inflicted, will reverse. Corneal injury reversibility is measured using Sodium Fluorescein (NaFl) stain to detect compromised epithelial barrier function. In this study, we examined the effects of five compounds on the cultured corneas: phosphate-buffered saline (PBS), 100% Ethanol (EtOH), 3% Sodium Dodecyl Sulfate (SDS), 1% Benzalkonium Chloride (BAK), and 10% Sodium Hydroxide (NaOH). Overall, the persistence of corneal effects between historical Draize rabbit eye data and PorCORA indicates a correlation coefficient of 0.98 (for the five compounds tested) and a correlation coefficient of 0.97 with the Draize modified maximal average score (MMAS). Finally, both fluorescence confocal microscopy and histopathology evidence demonstrates that the PorCORA and NaFl measurements are indicative of actual cellular and tissue damage. PorCORA shows promise as a potential non-animal replacement assay capable of predicting ocular damage reversibility. PMID:19735723

Piehl, Michelle; Gilotti, Albert; Donovan, Alison; DeGeorge, George; Cerven, Daniel

2010-02-01

68

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

PubMed

Infectious bursal disease virus (IBDV) can cause disease in chickens characterized by immunosuppression and high mortality. Currently, real-time RT-PCR has been used to quantitate virus-specific RNA and to better understand host response to infection. However, normalization of quantitative real-time RT-PCR is needed to a suitable internal control. We thus investigated the expression pattern of six chicken genes, including beta-actin, 28S rRNA, 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box-binding protein (TBP) and beta-2-microglobulin, in chicken embryo (CE) cell cultures following a 7-day IBDV infection. The CE cells were inoculated with various multiplicity of infection (MOI) of IBDV vaccine strain Bursine-2, the expression of genes was measured by quantitative real-time PCR-based on cDNA synthesized from either normalized (100 ng) or non-normalized (10 microl) total RNA. The results showed that beta-actin, 28S rRNA, 18S rRNA and GAPDH were the most constantly expressed genes, while TBP and beta-2-microglobulin were markedly induced during the infection course. Of these constant expressed genes, 28S rRNA and 18S rRNA are highly expressed; beta-actin intermediately expressed and GAPDH had a lower expression level in CE cell cultures. Also, beta-actin showed no significant variation in both normalized and non-normalized assays and virus dose-independent of inoculation, while other genes did. beta-Actin was further successfully used as an internal control to quantitate Bursine-2 virus-specific RNA load in CE cell cultures. Thus, beta-actin was suggested as a suitable internal control in studying gene expression as well as virus-specific RNA load in CE cell after IBDV infection. PMID:16159698

Li, Yi Ping; Bang, Dang D; Handberg, Kurt J; Jorgensen, Poul H; Zhang, Man Fu

2005-10-31

69

Assay of phagocytic cell functions.  

PubMed

The fundamental role of the immune system is recognition of the self from the non-self; in this way the principal functions of the immune system can be summarized as: resistance against the cells and foreign substances which are potentially damaging the tissues; identification of neoplastic cells to be destroyed. The cells which have this role are essentially lymphocyte, neutrophils and macrophages: extracellular and cellular humoral factors also play their role into the inflammatory process. In fact, we define the normal responses of phagocyte as the capacity of the specific phagocytic cell to respond to various stimuli and to migrate to the location of the damage. This complex cellular defense mechanism comprises several steps that can be summarized as following: opsonization of particles to be ingested, adhesion and aggregation of phagocytes to vascular endothelium, migration of phagocytes through the vessel walls, chemotaxis of phagocytes towards pathogenic agents, recognition of the particles/antigens by the phagocytes which subsequently adhere to their surface, ingestion of the particles with formation of a phagosome. This process is completed with the fusion of the phagosome with cellular granules (lysosomes) and formation of phagolysosomes, degranulation and release of the enzyme laden granules into the phagolysosome, lysis and killing of ingested particles and bacteria. It is clear from this schematic summary, that the response to pathogens can be very complex and each of the processes involved in the above described steps could be deranged leading to various pathological changes. We analyze the most classical and new methods to study the physiopathology of granulocytes, which are important for clinical diagnosis of phagocyte diseases or for phagocytic dysfunction in various syndromes and in neoplastic patients. PMID:8385468

Ricevuti, G; Mazzone, A; Fossati, G; Mazzucchelli, I; Cavigliano, P M; Pasotti, D; Notario, A

1993-02-01

70

Purified mouse mammary tumor and lymphoid cells in immune assays.  

PubMed

Tumor and lymphoid cell components from primary mammary adenocarcinomas of C3H/He mice were isolated simultaneously by velocity gradients. Viable tumor cells were obtained in sufficient numbers to test their in vivo and in vitro growth. Isolated tumor cells grew in 97% of inoculated syngeneic animals. In six assays with different tumors the effects of tumor-associated lymphoid cells (TAL) on in vivo tumor growth varied, enhancing in three and delaying in two experiments. Isolated tumor cells from animals with enhancing TAL grew faster in nonirradiated mice, whereas tumor cells from animals with inhibitory TAL grew better in irradiated animals. Isolated tumor cells also proliferated in cell culture, where they averaged 35% primary plating efficiency. Separated tumor cells were used in short-term 51Cr-release assays with TAL, tumor-bearer lymph node and spleen effectors. Cytotoxicity was detected in only five of 25 assays. In no case was there killing by lymphocyte populations from normal animals. In the present report we describe a technique for the isolation of viable tumor and lymphoid cells from murine adenocarcinomas that allows study of interactions between these populations from the original tumor-bearing host. PMID:6568874

Blazar, B A; Vanky, F; Klein, E

1984-01-01

71

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

72

[Ebola virus reproduction in cell cultures].  

PubMed

Ebola-Zaire virus production in Vero and BGM cells was studied. The CPE developed in both cell cultures. The cell monolayer destruction by 80-90% was seen at a low multiplicity of infection in 7-8 days after virus inoculation. An overlay composition was developed for virus titration using plaque assay. The plaque production was shown to be directly proportional to the virus dose. The curve of Ebola virus production in Vero cell culture fluid was determined. At a multiplicity of infection of 0.01 PFU/cell, the maximum virus titer of 10(6.4) PFU/ml was reached in 7 days postinfection. Specific antisera were generated by inoculation of guinea pigs. Indirect immunofluorescent assay was used for testing of virus-specific antigen and antibody. PMID:1279896

Titenko, A M; Novozhilov, S S; Andaev, E I; Borisova, T I; Kulikova, E V

1992-01-01

73

Endocytosis assays in intact and permeabilized cells.  

PubMed

Clathrin-coated pits and vesicles represent the major ports of entry into most eukaryotic cells. As well as performing housekeeping functions (e.g., allowing cells to take up essential nutrients), the endocytic pathway participates in a number of tissue-specific events such as synaptic-vesicle recycling, control of morphogen gradients during development, downregulation of receptor tyrosine kinases, and immune surveillance. To understand the role played by clathrin-mediated uptake, it is therefore essential to have robust endocytosis assays in intact cells. The clathrin-coated vesicle cycle requires a complicated interplay of proteins and lipids that is regulated in space and time. Reconstitution assays in permeabilized cells provide a powerful approach to understanding how this complex process is regulated. PMID:18228459

Osborne, Andrew; Flett, Alexander; Smythe, Elizabeth

2005-07-01

74

Plaque Assay of Rickettsiae in a Mammalian Cell Line  

PubMed Central

Clear-cut and repeatable plaque assays were obtained for three rickettsiae of the spotted fever group (Rickettsia rickettsi, R. conori, and R. montana) in Vero cells used in a manner similar to that for arboviruses. In addition, three typhus group agents (R. typhi, R. canada, R. prowazeki) induced plaques in these cells. In preliminary tests Coxiella burneti (Nine Mile strain) failed to produce plaques. Comparable results were obtained in plastic flasks and plastic culture trays incubated in ambient air with or without addition of N-2-hydroxyethyl-piperazine-N?-2-ethanesulfinic acid buffer. Larger and more well defined R. rickettsi plaques were produced when cultures were overlaid with Leibovitz (L15) medium than with either medium 199 or Eagle medium. Phosphate-buffered saline containing bovine plasma albumin (fraction V), in contrast to brain heart infusion broth, as a diluent for preparing inocula consistently permitted development of larger and more numerous plaques with three agents: R. rickettsi, R. conori, and R. montana. When R. rickettsi and R. typhi were assayed in parallel in primary chicken embryo cultures and Vero cells, comparable results were obtained, but with R. canada results in Vero cells were superior. In contrast, R. prowazeki produced inconsistent results in Vero cells. Images PMID:4208640

Cory, J.; Yunker, C. E.; Ormsbee, R. A.; Peacock, M.; Meibos, H.; Tallent, G.

1974-01-01

75

Enzyme?Linked Immunosorbent Assay Detection of Trichothecenes Produced by the Bioherbicide Myrothecium verrucaria in Cell Cultures, Extracts, and Plant Tissues  

Microsoft Academic Search

A rapid technique for trichothecene detection was needed in screening tests of the potential bioherbicide Myrothecium verrucaria (MV), in order to select strains, mutants, or formulations that were void of or that possessed low amounts of these undesirable mycotoxin compounds. Commercially available enzyme?linked immunosorbent assay (ELISA) plates for trichothecene detection, possessing cross?reactivity with several trichothecene mycotoxins (e.g., verrucarin A, and

Robert E. Hoagland; Mark A. Weaver; C. Douglas Boyette

2008-01-01

76

Expanding the Available Assays: Adapting and Validating In-Cell Westerns in Microfluidic Devices for Cell-Based Assays  

PubMed Central

Abstract Microfluidic methods for cellular studies can significantly reduce costs due to reduced reagent and biological specimen requirements compared with many traditional culture techniques. However, current types of readouts are limited and this lack of suitable readouts for microfluidic cultures has significantly hindered the application of microfluidics for cell-based assays. The In-Cell Western (ICW) technique uses quantitative immunocytochemistry and a laser scanner to provide an in situ measure of protein quantities in cells grown in microfluidic channels of arbitrary geometries. The use of ICWs in microfluidic channels was validated by a detailed comparison with current macroscale methods and shown to have excellent correlation. Transforming growth factor-?–induced epithelial-to-mesenchymal transition of an epithelial cell line was used as an example for further validation of the technique as a readout for soluble-factor-based assays performed in high-throughput microfluidic channels. The use of passive pumping for sample delivery and laser scanning for analysis opens the door to high-throughput quantitative microfluidic cell-based assays that integrate seamlessly with existing high-throughput infrastructure. PMID:20658945

Paguirigan, Amy L.; Puccinelli, John P.; Su, Xiaojing

2010-01-01

77

Urothelial cell culture.  

PubMed

This chapter reviews the use of urothelial cells as a means to enhance tissue regeneration and wound healing in urinary tract system. It addresses the properties of urothelial cells, including their role as a permeability barrier to protect underlying muscle tissue from the caustic effects of urine and as one of the main cell types, along with smooth muscle cells, that are used in urethral or bladder tissue engineering today. This description includes a general overview of various isolation techniques and culture methods that have been developed to improve urinary tract reconstruction in vivo and aid the characterization of growth factor expression in vitro. The chapter then describes various applications using urothelial cells, including production of multilayer urothelial sheets, tissue engineered bladder mucosa, tissue engineered urethra, and tissue engineered bladder. It also outlines the advantages of sandwich and layered coculture of these cells and the effects of epithelial-stromal cell interactions during tissue regeneration or wound healing processes in the urinary tract. PMID:24029928

Zhang, Yuanyuan; Atala, Anthony

2013-01-01

78

Selection and Separation of Viable Cells Based on a Cell-Lethal Assay  

PubMed Central

A method to select and separate viable cells based on the results of a cell-lethal assay was developed. Cells were plated on an array of culture sites with each site composed of closely spaced, releasable micropallets. Clonal colonies spanning multiple micropallets on individual culture sites were established within 72 h of plating. Adjacent sites were widely spaced with 100% of the colonies remaining sequestered on a single culture site during expansion. A laser-based method mechanically released a micropallet underlying a colony to segment the colony into two genetically identical colonies. One portion of the segmented colony was collected with 90% efficiency while viability of both fractions was 100%. The segmented colonies released from the array were fixed and subjected to immunofluorescence staining of intracellular phospho-ERK kinase to identify colonies that were highly resistant or sensitive to phorbol ester-induced activation of ERK. These resistant and sensitive cells were then matched to the corresponding viable colonies on the array. Sensitive and resistant colonies on the array were released and cultured. When these cultured cells were reanalyzed for phorbol ester-induced ERK activity, the cells retained the sensitive or resistant phenotype of the originally screened subcolony. Thus cells were separated and collected based using the result of a cell-lethal assay as selection criteria. These microarrays enabling clonal colony segmentation permitted sampling and manipulation of the colonies at very early times and at small cell numbers to reduce reagent, time and manpower requirements. PMID:21142138

Xu, Wei; Herman, Annadele; Phillips, Colleen; Pai, Jeng-Hao; Sims, Christopher E.; Allbritton, Nancy L.

2010-01-01

79

Cell culture's spider silk road.  

PubMed

A number of synthetic and natural materials have been tried in cell culture and tissue engineering applications in recent years. Now Jeffrey Perkel takes a look at one new culture component that might surprise you-spider silk. PMID:24924388

Perkel, Jeffrey

2014-06-01

80

Quantitative determination of urea concentrations in cell culture medium  

PubMed Central

Urea is the major nitrogenous end product of protein metabolism in mammals. Here, we describe a quantitative, sensitive method for urea determination using a modified Jung reagent. This assay is specific for urea and is unaffected by ammonia, a common interferent in tissue and cell cultures. We demonstrate that this convenient colorimetric microplate-based, room temperature assay can be applied to determine urea synthesis in cell culture. PMID:19448747

Zawada, Robert J.X.; Kwan, Peggy; Olszewski, Kellen L.; Llinas, Manuel; Huang, Shu-Gui

2009-01-01

81

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

82

Illuminations: A Cell Notes Publication | January 2014 32 Illuminations: A Cell Notes Publication | January 2014 How to Choose a Cell Health Assay  

E-print Network

health assay needed. There are cell health assays available to specifically detect the number of living cells, the number of dead cells, and for assessing stress response mechanisms or pathways that may morphology or measurement of markers using reagents compatible with living cultures (real-time analysis

Cai, Long

83

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

84

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

PubMed Central

Three commonly used isolates of murine prions, 79A, 139A, and RML, were derived from the so-called Chandler isolate, which was obtained by propagating prions from scrapie-infected goat brain in mice. RML is widely believed to be identical with 139A; however, using the extended cell panel assay (ECPA), we here show that 139A and RML isolates are distinct, while 79A and RML could not be distinguished. We undertook to clone 79A and 139A prions by endpoint dilution in murine neuroblastoma-derived PK1 cells. Cloned 79A prions, when returned to mouse brain, were unchanged and indistinguishable from RML by ECPA. However, 139A-derived clones, when returned to brain, yielded prions distinct from 139A and similar to 79A and RML. Thus, when 139A prions were transferred to PK1 cells, 79A/RML-like prions, either present as a minor component in the brain 139A population or generated by mutation in the cells, were selected and, after being returned to brain, were the major if not only component of the population. PMID:22379091

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

2012-01-01

85

Orosphere Assay: A method for propagation of head and neck cancer stem cells  

PubMed Central

Background Recent evidence suggests that head and neck squamous cell carcinomas (HNSCC) harbor a small sub-population of highly tumorigenic cells, named cancer stem cells. A limiting factor in cancer stem cell research is the intrinsic difficulty of expanding cells in an undifferentiated state in vitro. Methods Here, we describe the development of the orosphere assay, a method for the study of putative head and neck cancer stem cells. An orosphere is defined as a non-adherent colony of cells sorted from primary HNSCC or from HNSCC cell lines and cultured in 3-D soft agar or ultra-low attachment plates. Aldehyde dehydrogenase (ALDH) activity and CD44 expression were used here as stem cell markers. Results This assay allowed for the propagation of head and neck cancer cells that retained stemness and self-renewal. Conclusion The orosphere assay is well suited for studies designed to understand the pathobiology of head and neck cancer stem cells. PMID:22791367

Krishnamurthy, Sudha; Nor, Jacques E.

2014-01-01

86

Plant cell suspension culture rheology.  

PubMed

The results of rheological measurements on 10 different plant cell suspension cultures are presented. Nicotiana tabacum (tobacco) suspension cultures grown in serial batch subculture display high viscosity and power law rheology. This "undesirable" rheology is shown to be a result of elongated cell morphology. The rheology of Papaver somniferum (poppy) cell suspensions is quite different; poppy suspensions behave as Newtonian fluids and have relatively low viscosity (less than 15 cP) at fresh cell densities up to 250 g/L. This flow behavior can be attributed to a lack of elongation in batch-grown poppy cells. A simple correlation for the viscosity as a function of cell density is developed for poppy suspensions up to 300 g fresh weight (FW)/L. It is shown that tobacco cells do not elongate when grown in semicontinuous culture (daily media replacement). These semicontinuously cultured cells have rheological behavior that is indistinguishable from that of poppy, further confirming the dependence of rheology on plant cell morphology. The rheology of a wide variety of other plant suspensions at 200 g FW/L is presented. Most cell suspensions, including soybean, cotton, bindweed, and potato, display low viscosities similar to poppy suspensions. Only carrot and atriplex exhibit slight pseudoplastic behavior which corresponded to a slight degree of cellular elongation for these cultures. This demonstrates that complex rheology associated with elongated cell morphology is much less common than low-viscosity Newtonian behavior. High viscosity in plant cell culture is therefore not an intrinsic characteristic of plant cells but, instead, is a result of the ability to grow cultures to extremely high cell densities due to low biological oxygen demand. PMID:18613057

Curtis, W R; Emery, A H

1993-08-01

87

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

88

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.

89

Type and Subtype-Specific Detection of Influenza Viruses in Clinical Specimens by Rapid Culture Assay  

Microsoft Academic Search

A rapid culture assay which allows for the simultaneous typing and subtyping of currently circulating influenza A(H1N1), A(H3N2), and B viruses in clinical specimens was developed. Pools of monoclonal anti- bodies(MAbs)againstinfluenzaAandBvirusesandMAbsHA1-71andHA2-76,obtainedbyimmunizingmice withthedenaturedhemagglutininsubfragmentsHA1andHA2ofinfluenzavirusA\\/Victoria\\/3\\/75,wereusedfor immunoperoxidase staining of antigens in infected MDCK cells. MAb HA1-71 reacted exclusively with influ- enza A viruses of the H3 subtype, while MAb HA2-76 reacted with subtypes H1,

THEDI ZIEGLER; HENRIETTA HALL; ALICIA SANCHEZ-FAUQUIER; WILLIAM C. GAMBLE; J. COX

1995-01-01

90

The molecular bacterial load assay replaces solid culture for measuring early bactericidal response to antituberculosis treatment.  

PubMed

We evaluated the use of the molecular bacterial load (MBL) assay, for measuring viable Mycobacterium tuberculosis in sputum, in comparison with solid agar and liquid culture. The MBL assay provides early information on the rate of decline in bacterial load and has technical advantages over culture in either form. PMID:24871215

Honeyborne, Isobella; Mtafya, Bariki; Phillips, Patrick P J; Hoelscher, Michael; Ntinginya, Elias N; Kohlenberg, Anke; Rachow, Andrea; Rojas-Ponce, Gabriel; McHugh, Timothy D; Heinrich, Norbert

2014-08-01

91

A simple method for the quantitation of the stem cells derived from human exfoliated deciduous teeth using a luminescent cell viability assay.  

PubMed

Stem cells from human exfoliated deciduous teeth are a population of highly proliferative postnatal stem cells and have been characterized as multipotent stem cells. In this study we developed a fast and sensitive method for stem cells derived from human exfoliated deciduous teeth count, using a luminescent viability assay. We isolated stem cells from normal exfoliated deciduous teeth using collagenase/dispase digestions solutions. Separated stem cells were placed in opaque-walled multiwall plates in culture alpha Modified Eagle's Medium. For dental pulp stem cells quantitation we used a simple method for determining the number of viable cells based on ATP concentration. Cells attached to the bottom of the multiwall plates were counted with the luminescent assay and were cultured for mesenchymal markers expression. Moreover cells attached to the bottom of the multiwall plates were directed toward the osteogenic, adipogenic, lineages at the respective passages. Flow cytometry was used for immunophenotyping of cultured dental stem cells from exfoliated deciduous teeth. Cells that were counted with the luminescent assay, after culture formed fibroblastic morphology and were expressed the mesenchymal stem cell markers CD29, CD105, CD146, CD44. There was a correlation between the number of cells plated for culture and the number of mesenchymal stem cells after culture. Osteogenic and adipogenic differentiation of the cells counted with the luminescent assay was performed. The luminescent signal of viable mesenchymal dental stem cells isolated from dental pulp of exfoliated teeth represents an ideal method for mesenchymal stem cells count before culturing. PMID:24379108

Tsagias, Nikos; Koliakos, Kokkona-Kouzi; Spyridopoulos, Themistoklis; Pitiakoudis, Michail; Koliakos, Eleni; Korpeti, Ioanna; Koliakos, George

2014-09-01

92

Translation of a tumor microenvironment mimicking 3D tumor growth co-culture assay platform to high-content screening.  

PubMed

For drug discovery, cell-based assays are becoming increasingly complex to mimic more realistically the nature of biological processes and their diversifications in diseases. Multicellular co-cultures embedded in a three-dimensional (3D) matrix have been explored in oncology to more closely approximate the physiology of the human tumor microenvironment. High-content analysis is the ideal technology to characterize these complex biological systems, although running such complex assays at higher throughput is a major endeavor. Here, we report on adapting a 3D tumor co-culture growth assay to automated microscopy, and we compare various imaging platforms (confocal vs. nonconfocal) with correlating automated image analysis solutions to identify optimal conditions and settings for future larger scaled screening campaigns. The optimized protocol has been validated in repeated runs where established anticancer drugs have been evaluated for performance in this innovative assay. PMID:22923784

Krausz, Eberhard; de Hoogt, Ronald; Gustin, Emmanuel; Cornelissen, Frans; Grand-Perret, Thierry; Janssen, Lut; Vloemans, Nele; Wuyts, Dirk; Frans, Sandy; Axel, Amy; Peeters, Pieter Johan; Hall, Brett; Cik, Miroslav

2013-01-01

93

Immobilized MWCNT support osteogenic cell culture.  

PubMed

The broad use of versatile, strong, lightweight multi-walled carbon nanotubes (MWCNT) for use in biomaterial applications is tempered by ongoing debate about their safety. Recent reports suggest that factors such as their diameter and surface coating affect their function and cytotoxicity. The cell culture surfaces used in the current study were made of MWCNT immobilized in a high-density polyethylene substrate, differentiating it from most studies of MWCNT cytotoxicity. The purity, chemical functionalization, and immobilization of MWCNT were evaluated to elucidate their effect on MWCNT behavior relative to controls. While purity was found not to be significant in determining the behavior of cells on MWCNT relative to standard controls, the presence of carboxyl functional groups was generally associated with reduced cell metabolic activity, proliferation, and differentiation as measured using the MTS assay, nucleic acid incorporation, and alkaline phosphatase expression, respectively. This study demonstrates that the culture of osteogenic cells on surfaces made of nonfunctionalized and immobilized MWCNT is associated with a level of cell growth and differentiation comparable to those of standard tissue culture controls. PMID:23471503

Emohare, Osa; Rushton, Neil

2013-06-01

94

Novel cultured porcine corneal irritancy assay with reversibility endpoint  

Microsoft Academic Search

Several alternative assays exist to assess ocular irritancy without the use of live animals. However, these assays cannot address ocular injury reversibility. Reversibility is an issue critical to regulatory authorities and manufactures of commercial products, as ocular irritation caused by misuse or accidental exposure to a product may cause irreversible eye damage. Here we report the development and initial characterization

Michelle Piehl; Albert Gilotti; Alison Donovan; George DeGeorge; Daniel Cerven

2010-01-01

95

The culture of limbal epithelial cells.  

PubMed

The transplantation of cultured limbal epithelial cells (LEC) has since its first application in 1997 emerged as a promising technique for treating limbal stem cell deficiency. The culture methods hitherto used vary with respect to preparation of the harvested tissue, choice of culture medium, culture time, culture substrates, and supplementary techniques. In this chapter, we describe a procedure for establishing human LEC cultures using a feeder-free explant culture technique with human amniotic membrane (AM) as the culture substrate. PMID:23690008

Utheim, Tor Paaske; Lyberg, Torstein; Ræder, Sten

2013-01-01

96

Circulating Tumor Cell Assay Quality Control and Trouble Shooting Guide  

Cancer.gov

1.1 1.2 Version: July 2010 1.3 Circulating Tumor Cell Assay Quality Control and Trouble Shooting Guide LHTP003.8.1 ?H2AX IMMUNOFLUORESCENCE ASSAY FOR CIRCULATING TUMOR CELLS USING THE CELLSEARCH SYSTEM LHTP003.8.1.1 ?H2AX IMMUNOFLUORESCENCE

97

Multiwell cell culture plate format with integrated microfluidic perfusion system  

NASA Astrophysics Data System (ADS)

A new cell culture analog has been developed. It is based on the standard multiwell cell culture plate format but it provides perfused three-dimensional cell culture capability. The new capability is achieved by integrating microfluidic valves and pumps into the plate. The system provides a means to conduct high throughput assays for target validation and predictive toxicology in the drug discovery and development process. It can be also used for evaluation of long-term exposure to drugs or environmental agents or as a model to study viral hepatitis, cancer metastasis, and other diseases and pathological conditions.

Domansky, Karel; Inman, Walker; Serdy, Jim; Griffith, Linda G.

2006-01-01

98

Rapid, Specific Detection of Alphaviruses from Tissue Cultures Using a Replicon-Defective Reporter Gene Assay  

PubMed Central

We established a rapid, specific technique for detecting alphaviruses using a replicon-defective reporter gene assay derived from the Sindbis virus XJ-160. The pVaXJ expression vector containing the XJ-160 genome was engineered to form the expression vectors pVaXJ-EGFP expressing enhanced green fluorescence protein (EGFP) or pVaXJ-GLuc expressing Gaussia luciferase (GLuc). The replicon-defective reporter plasmids pVaXJ-EGFP?nsp4 and pVaXJ-GLuc?nsp4 were constructed by deleting 1139 bp in the non-structural protein 4 (nsP4) gene. The deletion in the nsP4 gene prevented the defective replicons from replicating and expressing reporter genes in transfected BHK-21 cells. However, when these transfected cells were infected with an alphavirus, the non-structural proteins expressed by the alphavirus could act on the defective replicons in trans and induce the expression of the reporter genes. The replicon-defective plasmids were used to visualize the presence of alphavirus qualitatively or detect it quantitatively. Specificity tests showed that this assay could detect a variety of alphaviruses from tissue cultures, while other RNA viruses, such as Japanese encephalitis virus and Tahyna virus, gave negative results with this system. Sensitivity tests showed that the limit of detection (LOD) of this replicon-defective assay is between 1 and 10 PFU for Sindbis viruses. These results indicate that, with the help of the replicon-defective alphavirus detection technique, we can specifically, sensitively, and rapidly detect alphaviruses in tissue cultures. The detection technique constructed here may be well suited for use in clinical examination and epidemiological surveillance, as well as for rapid screening of potential viral biological warfare agents. PMID:22427930

Wang, Huanqin; Li, Jiandong; Zhang, Quanfu; He, Ying; Li, Jia; Fu, Juanjuan; Li, Dexin; Liang, Guodong

2012-01-01

99

Basic Laboratory Techniques in Cell Culture.  

National Technical Information Service (NTIS)

This is a reference and instructional manual for the Centers for Disease Control (CDC) Course Number 8270-C, 'Basic Laboratory Techniques in Cell Culture.' It covers pertinent cell biology, cell culture terminology and definitions, the principles and meth...

B. R. Bird, F. T. Forrester

1981-01-01

100

Versatile, Fully Automated, Microfluidic Cell Culture System  

E-print Network

on a microfluidic chip that creates arbitrary culture media formulations in 96 independent culture chambersVersatile, 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

Chen, Christopher S.

101

INFLUENCE OF TEMPERATURE ON AN ESTROGEN-RESPONSIVE RAINBOW TROUT CELL TRANSFECTION ASSAY  

EPA Science Inventory

One uncertainty in extrapolating estrogenic effects in mammalian systems to those in fish and wildlife is the influence that temperature has on these effects. A reporter gene assay in cultured rainbow trout cell lines was used to determine the influence of temperature on the exp...

102

Keratinocyte Stem Cell Assays: An Evolving Science  

Microsoft Academic Search

Although the existence of epithelial stem cells in the skin has been known for some decades from cell kinetic studies performed in vivo, attempts to prospectively isolate these cells for further biological characterization have been made possible relatively recently facilitated by the availability of antibodies that detect cell surface markers on epidermal cells. Elegant gene marking studies in vivo have

Pritinder Kaur; Amy Li; Richard Redvers; Ivan Bertoncello

2004-01-01

103

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

104

Detection of campylobacter in gastroenteritis: comparison of direct PCR assay of faecal samples with selective culture.  

PubMed

The prevalence of campylobacter gastroenteritis has been estimated by bacterial isolation using selective culture. However, there is evidence that certain species and strains are not recovered on selective agars. We have therefore compared direct PCR assays of faecal samples with campylobacter culture, and explored the potential of PCR for simultaneous detection and identification to the species level. Two hundred unselected faecal samples from cases of acute gastroenteritis were cultured on modified charcoal cefoperazone deoxycholate agar and subjected to DNA extraction and PCR assay. Culture on CCDA indicated that 16 of the 200 samples contained 'Campylobacter spp.'. By contrast, PCR assays detected campylobacters in 19 of the 200 samples, including 15 of the culture-positive samples, and further identified them as: C. jejuni (16), C. coli (2) and C. hyointestinalis (1). These results show that PCR offers a different perspective on the incidence and identity of campylobacters in human gastroenteritis. PMID:10030703

Lawson, A J; Shafi, M S; Pathak, K; Stanley, J

1998-12-01

105

Production of tropane alkaloids in cultured cells of Hyoscyamus niger.  

PubMed

Tests for calluses rich in tropane alkaloids were made with newly induced calluses of Atropa belladonna, Datura stramonium and Hyoscyamus niger. Only calluses of H. niger gave an alkaloid-positive test.A Hyoscyamus cell line had the highest total alkaloid content of all the calluses screened by the cell-squash alkaloid assay. Both hyoscyamine and scopolamine were identified in the cultured cells of this line by TLC, GLC and GC-MS. PMID:24259019

Yamada, Y; Hashimoto, T

1982-04-01

106

High-throughput screening of small molecules in miniaturized mammalian cell-based assays involving post-translational  

E-print Network

High-throughput screening of small molecules in miniaturized mammalian cell-based assays involving systems using small molecules, rather than mutations, as the source of gene- product alterations of small molecules in nanoliter to microliter culture volumes. We refer to this assay format as a `cytoblot

Stockwell, Brent R.

107

A microfluidic device for uniform-sized cell spheroids formation, culture, harvesting and flow cytometry analysis  

PubMed Central

Culture of cells as three-dimensional (3D) aggregates, named spheroids, possesses great potential to improve in vitro cell models for basic biomedical research. However, such cell spheroid models are often complicated, cumbersome, and expensive compared to conventional Petri-dish cell cultures. In this work, we developed a simple microfluidic device for cell spheroid formation, culture, and harvesting. Using this device, cells could form uniformly sized spheroids due to strong cell–cell interactions and the spatial confinement of microfluidic culture chambers. We demonstrated cell spheroid formation and culture in the designed devices using embryonic stem cells, carcinoma cells, and fibroblasts. We further scaled up the device capable of simultaneously forming and culturing 5000 spheroids in a single chip. Finally, we demonstrated harvesting of the cultured spheroids from the device with a simple setup. The harvested spheroids possess great integrity, and the cells can be exploited for further flow cytometry assays due to the ample cell numbers. PMID:24396525

Patra, Bishnubrata; Chen, Ying-Hua; Peng, Chien-Chung; Lin, Shiang-Chi; Lee, Chau-Hwang; Tung, Yi-Chung

2013-01-01

108

21 CFR 864.7100 - Red blood cell enzyme assay.  

Code of Federal Regulations, 2012 CFR

... FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification....

2012-04-01

109

21 CFR 864.7100 - Red blood cell enzyme assay.  

... FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification....

2014-04-01

110

21 CFR 864.7100 - Red blood cell enzyme assay.  

Code of Federal Regulations, 2011 CFR

... FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification....

2011-04-01

111

Improved plaque assays for Rickettsia prowazekii in Vero 76 cells.  

PubMed Central

Typhus group rickettsiae, including Rickettsia prowazekii and R. typhi, produce visible plaques on primary chick embryo fibroblasts and low-passage mouse embryo fibroblasts but do not form reproducible plaques on continuous cell culture lines. We tested medium overlay modifications for plaque formation of typhus group rickettsiae on the continuous fibroblast cell line Vero76. A procedure involving primary overlay with medium at pH 6.8, which was followed 2 to 3 days later with secondary overlay at neutral pH containing 1 microgram of emetine per ml and 20 micrograms of NaF per ml, resulted in visible plaques at 7 to 10 days postinfection. A single-step procedure involving overlay with medium containing 50 ng of dextran sulfate per ml also resulted in plaque formation within 8 days postinfection. These assays represent reproducible and inexpensive methods for evaluating the infectious titers of typhus group rickettsiae, cloning single plaque isolates, and testing the susceptibilities of rickettsiae to antibiotics. PMID:8818887

Policastro, P F; Peacock, M G; Hackstadt, T

1996-01-01

112

A cell-based reporter assay for screening for EcR agonist/antagonist activity of natural ecdysteroids in Lepidoptera (Bm5) and Diptera (S2) cell cultures, followed by modeling of ecdysteroid-EcR interactions and normal mode analysis.  

PubMed

Ecdysteroid signal transduction is a key process in insect development and therefore an important target for insecticide development. We employed an in vitro cell-based reporter bioassay for the screening of potential ecdysone receptor (EcR) agonistic and antagonistic compounds. Natural ecdysteroids were assayed with ecdysteroid-responsive cell line cultures that were transiently transfected with the reporter plasmid ERE-b.act.luc. We used the dipteran Schneider S2 cells of Drosophila melanogaster and the lepidopteran Bm5 cells of Bombyx mori, representing important pest insects in medicine and agriculture. Measurements showed an EcR agonistic activity only for cyasterone both in S2 (EC50=3.3?M) and Bm5 cells (EC50=5.3?M), which was low compared to that of the commercial dibenzoylhydrazine-based insecticide tebufenozide (EC50=0.71?M and 0.00089?M, respectively). Interestingly, a strong antagonistic activity was found for castasterone in S2 cells with an IC50 of 0.039?M; in Bm5 cells this effect only became visible at much higher concentrations (IC50=18?M). To gain more insight in the EcR interaction, three-dimensional modeling of dipteran and lepidopteran EcR-LBD was performed. In conclusion, we showed that the EcR cell-based reporter bioassay tested here is a useful and practical tool for the screening of candidate EcR agonists and antagonists. The docking experiments as well as the normal mode analysis provided evidence that the antagonist activity of castasterone may be through direct binding with the receptor with specific changes in protein flexibility. The search for new ecdysteroid-like compounds may be particularly relevant for dipterans because the activity of dibenzoylhydrazines appears to be correlated with an extension of the EcR-LBD binding pocket that is prominent in lepidopteran receptors but less so in the modeled dipteran structure. PMID:24267692

Zotti, Moisés J; De Geyter, Ellen; Swevers, Luc; Braz, Antônio S K; Scott, Luis P B; Rougé, Pierre; Coll, Josep; Grutzmacher, Anderson D; Lenardão, Eder J; Smagghe, Guy

2013-11-01

113

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...

114

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

115

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

116

Ligase chain reaction assay for human mutations: the Sickle Cell by LCR assay.  

PubMed

We can detect the beta-globin gene sickle cell mutation by using an assay based on the ligase chain reaction. The simultaneous amplification of the human growth hormone gene in the same reaction serves as a control for the amount of template DNA or amplification efficiency. Ligation products, which are biotinylated at one end and tagged with an arbitrary "tail" sequence at the other, are captured by hybridization to "tail"-complementary oligonucleotides immobilized on polystyrene microwells. The captured ligation products are detected colorimetrically by use of streptavidin-alkaline phosphatase conjugate. In a study of 24 subjects, the assay unequivocally discriminated among normal, carrier, and sickle cell genotypes. PMID:8990220

Reyes, A A; Carrera, P; Cardillo, E; Ugozzoli, L; Lowery, J D; Lin, C I; Go, M; Ferrari, M; Wallace, R B

1997-01-01

117

A Simple High-Content Cell Cycle Assay Reveals Frequent Discrepancies between Cell Number and ATP and MTS Proliferation Assays  

PubMed Central

In order to efficiently characterize both antiproliferative potency and mechanism of action of small molecules targeting the cell cycle, we developed a high-throughput image-based assay to determine cell number and cell cycle phase distribution. Using this we profiled the effects of experimental and approved anti-cancer agents with a range mechanisms of action on a set of cell lines, comparing direct cell counting versus two metabolism-based cell viability/proliferation assay formats, ATP-dependent bioluminescence, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) reduction, and a whole-well DNA-binding dye fluorescence assay. We show that, depending on compound mechanisms of action, the metabolism-based proxy assays are frequently prone to 1) significant underestimation of compound potency and efficacy, and 2) non-monotonic dose-response curves due to concentration-dependent phenotypic ‘switching’. In particular, potency and efficacy of DNA synthesis-targeting agents such as gemcitabine and etoposide could be profoundly underestimated by ATP and MTS-reduction assays. In the same image-based assay we showed that drug-induced increases in ATP content were associated with increased cell size and proportionate increases in mitochondrial content and respiratory flux concomitant with cell cycle arrest. Therefore, differences in compound mechanism of action and cell line-specific responses can yield significantly misleading results when using ATP or tetrazolium-reduction assays as a proxy for cell number when screening compounds for antiproliferative activity or profiling panels of cell lines for drug sensitivity. PMID:23691072

Chan, Grace Ka Yan; Kleinheinz, Tracy L.; Peterson, David; Moffat, John G.

2013-01-01

118

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

119

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

120

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

121

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

122

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

123

A comparison of binax™ now® to viral culture and direct fluorescent assay testing for respiratory syncytial virus  

Microsoft Academic Search

The Binax™ NOW® immunochromatographic assay for respiratory syncytial virus was prospectively compared with direct fluorescent assay and viral culture at Primary Children's Medical Center, Salt Lake City, Utah during February 2003. Three hundred ten patient specimens were collected for testing, of which 102 specimens were positive for respiratory syncytial virus by the reference tests, direct immunofluorescence assay (DFA), and culture

Wade K Aldous; Kris Gerber; Edward W Taggart; Joshua Thomas; David Tidwell; Judy A Daly

2004-01-01

124

A one-day, dispense-only IP-One HTRF assay for high-throughput screening of Galphaq protein-coupled receptors: towards cells as reagents.  

PubMed

Abstract: Compared to biochemical high-throughput screening (HTS) assays, cell-based functional assays are generally thought to be more time consuming and complex because of additional efforts for running continuous cell cultures as well as the numerous assay steps when transferring media and compounds. A common strategy to compensate the anticipated reduction in overall throughput is to implement highly automated cell culture and screening systems. However, such systems require substantial investments in sophisticated hardware and highly specialized personnel. In trying to set up alternatives to increasing throughput in functional cell-based screening, we combined several approaches. By using (1) cryopreserved cell aliquots instead of continuous cell culture, (2) cells in suspension instead of adherent cells, and (3) "ready-to-screen" assay plates with nanoliter aliquots of test compounds, an assay procedure was developed that very much resembles a standard biochemical, enzymatic assay comprising only a few dispense steps. Chinese hamster ovary cells stably overexpressing a Galphaq-coupled receptor were used as a model system to measure receptor activation by detection of intracellular D-myo-inositol 1-phosphate with the help of homogeneous time-resolved fluorescence (HTRF, CISbio International, Bagnols-sur-Cèze, France). Initially established in 384-well adherent cell format, the assay was successfully transferred to 1,536-well format. The assay quality was sufficient to run HTS campaigns in both formats with good Z'-factors and excellent reproducibility of antagonists. Subsequently, the assay procedure was optimized for usage of suspension cells. The influences of cell culture media, plate type, cell number, and incubation time were assessed. Finally, the suspension cell assay was applied to pharmacological characterization of a small molecule antagonist by Schild plot analysis. Our data demonstrate not only the application of the IP-One HTRF assay (CISbio International) for HTS in a high-density format, but furthermore the successful use of cryopreserved and suspension cells in a one-day functional cell-based assay. PMID:18315499

Bergsdorf, Christian; Kropp-Goerkis, Carmen; Kaehler, Irene; Ketscher, Lars; Boemer, Ulf; Parczyk, Karsten; Bader, Benjamin

2008-02-01

125

A microfluidic system for automatic cell culture  

NASA Astrophysics Data System (ADS)

This study presents a new chip capable of automating the cell culture process by using microfluidic technology. This microfluidic cell culture system comprising microheaters, a micro temperature sensor, micropumps, microvalves, microchannels, a cell culture area and several reservoirs was fabricated by using micro-electro-mechanical-systems' fabrication processes. Traditional manual cell culture processes can be performed on this chip. A uni-directional pneumatic micropump was developed to transport the culture reagents and constraint the solutions to flow only in one direction, safeguarding the entire culture process from contamination. A new micro check valve was also used to prevent the culture solutions from flowing back into the microchannels. The microheaters and the micro temperature sensor were used to maintain a constant temperature during the cell culturing process. The pH value suitable for cell growth was also regulated during the cell culture process. A typical cell culturing process for human lung cancer cells (A549) was successfully performed to demonstrate the capability of the developed microfluidic system. This automatic cell culturing system can be eventually integrated with subsequent microfluidic modules for cell purification, collection, counting and lysis to form a cell-based micro-total-analysis system. Preliminary results have been presented in The Asia-Pacific Conference of Transducers and Micro-Nano Technology (APCOT), 25-28 June 2006

Huang, Chun-Wei; Lee, Gwo-Bin

2007-07-01

126

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

PubMed

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

2014-03-01

127

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

128

Chromosome preparation from cultured cells.  

PubMed

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births(1,2), 60-80% of all miscarriages(3,4), 10% of stillbirths(2,5), 13% of individuals with congenital heart disease(6), 3-6% of infertility cases(2), and in many patients with developmental delay and birth defects(7). Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance(8,9). Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents(10-13). Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy's fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)(14,15). PMID:24513647

Howe, Bradley; Umrigar, Ayesha; Tsien, Fern

2014-01-01

129

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

2014-11-10

130

Normal and leukemic human stem cells assayed in SCID mice  

Microsoft Academic Search

Understanding the processes that regulate the developmental program of normal stem cells and those that initiate proliferative diseases such as leukemia remains one of the major challenges in biology. Progress to address these major questions in the human hematopoietic system have been hampered, until recently, by the lack of in-vivo assays for normal and leukemic stem cells. The recent development

John E. Dick

1996-01-01

131

Dynamized Preparations in Cell Culture  

PubMed Central

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

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

2009-01-01

132

LANTHANUM IN HEART CELL CULTURE  

PubMed Central

Correlation of the localization of La+++ with its effects on Ca++ exchange in cultured rat heart cells is examined with the use of a recently developed technique. 75% of cellular Ca++ is exchangeable and is completely accounted for by two kinetically defined phases. The rapidly exchangeable phase has a t ½ = 1.15 min and accounts for 1 1 mmoles Ca++/kg wet cells or 43% of the exchangeable Ca++ (cells perfused with [Ca++]o = 1 mM) Phase 2 has a t ½ = 19.2 min and accounts for 1.5 mmoles Ca++/kg wet cells or 57% of the exchangeable Ca++. 0.5 mM [La+++]o displaces 0 52 mmoles Ca++/kg wet cells—all from phase 1—and almost completely abolishes subsequent Ca++ influx and efflux The presence of La+++ in the washout converts the washout pattern to a single phase system with a t ½ = 124 min. The effects upon Ca++ exchange are coincident with abolition of contractile tension but regenerative depolarization of the tissue is maintained Electron microscope localization of the La+++ places it exclusively in the external lamina or basement membrane of the cells. The study indicates that negatively charged sites in the basement membrane play a crucial role in the E-C coupling process in heart muscle PMID:5044754

Langer, G. A.; Frank, J. S.

1972-01-01

133

The glycophorin A assay for somatic cell mutations in humans  

SciTech Connect

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

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

1989-08-18

134

One mouse, two cultures: isolation and culture of adult neural stem cells from the two neurogenic zones of individual mice.  

PubMed

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

135

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

136

Chemical potential of Aphelandra sp. cell cultures  

Microsoft Academic Search

Six different callus lines and three different suspension culture lines were established from plants of two Aphelandra species (Acanthaceae). All established lines were analyzed for secondary metabolite accumulation. A discrepancy between secondary\\u000a metabolites accumulated in the plants and in the cell cultures could be observed. All established Aphelandrasp. cell cultures produced verbascoside (acteoside) as the major extractable metabolite. Time course

Lenka Nezbedová; Manfred Hesse; Jaroslav Dušek; Christa Werner

1999-01-01

137

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

PubMed Central

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

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

2006-01-01

138

Catechin production in cultured Polygonum hydropiper cells  

Microsoft Academic Search

Callus and suspension-cultured cells were induced from hypocotyls of Polygonum hydropiper seedlings. Both the callus and suspension-cultured cells produced mainly (+)-catechin accompanied by (?)-epicatechin and (?)-epicatechin-3-O-gallate. The (+)-catechin production of suspension-cultured cells increased with cell growth and reached the maximal value (29.0mgg?1 dry wt) after 6days from the start of subculture. This is the highest value of (+)-catechin content among

Kanji Ono; Mayumi Nakao; Masao Toyota; Yoshimi Terashi; Masashi Yamada; Tetsuya Kohno; Yoshinori Asakawa

1998-01-01

139

Carrot Embryogenesis from Frozen Cultured Cells  

Microsoft Academic Search

MANY plant tissue cultures change in growth rate, chromosome cytology and morphogenic potential during repeated subculture1-3. Controlled freezing and low temperature storage of cultured plant cells might enable the characters of newly initiated cultures to be preserved. Cell preservation at the temperature of liquid N2 (-196° C) has been successful with animal cells4 and preliminary work with plant cells has

K. K. Nag

1973-01-01

140

Calcium Signaling Mechanisms Mediate Clock-Controlled ATP Gliotransmission among Immortalized Rat SCN2.2 Cell Cultures  

E-print Network

of the Luciferin/Luciferase Assay........................................ 27 2 Stimulation of SCN2.2 Cell Cultures with Glass Microbeads Led to Detectable ATP Chemiluminescence......................................................... 28 3... of the Luciferin/Luciferase Assay........................................ 27 2 Stimulation of SCN2.2 Cell Cultures with Glass Microbeads Led to Detectable ATP Chemiluminescence......................................................... 28 3...

Burkeen, Jeffrey Franklin

2010-10-12

141

Ensemble Analysis of Angiogenic Growth in Three-Dimensional Microfluidic Cell Cultures  

E-print Network

We demonstrate ensemble three-dimensional cell cultures and quantitative analysis of angiogenic growth from uniform endothelial monolayers. Our approach combines two key elements: a micro-fluidic assay that enables ...

Farahat, Waleed A.

142

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

143

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

144

Teratological research using in vitro systems. IV. Cells in culture.  

PubMed Central

Several in vitro cellular systems designed to screen agents for teratogenic potential are described in this report. These assays were selected from a review of literature published through the spring of 1986 that generated over 100 references on teratological research using cell-based systems. Some of the assays have a broader application than others, but most require confirmation by one or more additional complementary tests because of the specificity of the teratogenic mechanism the assays are investigating. Included are systems that use analysis of tumor cell attachment; intercellular communication; growth of human embryonic palatal mesenchyme cells; progesterone production in porcine granulosa cells; differentiation of embryonic neural crest, limb bud, midbrain, and Drosophila cells; and differentiation of tumor cells. Because of the dynamic nature of cell culture work, the group of assays listed here should not be viewed as encompassing all cell systems of value with regard to teratogenicity testing; instead, the list represents several of the more prominent systems now being evaluated by the scientific community. PMID:3113933

Welsh, J J

1987-01-01

145

Primary cell cultures from Drosophila gastrula embryos.  

PubMed

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells. PMID:21403631

Perrimon, Norbert; Zirin, Jonathan; Bai, Jianwu

2011-01-01

146

Label-free cell-based dynamic mass redistribution assays.  

PubMed

Label-free cell-based assays offer a powerful approach to drug discovery and compound profiling for endogenously expressed receptors in a variety of cell types, including primary and stem cells. Dynamic mass redistribution (DMR) responses in whole cells following receptor stimulation provide phenotypic activity profiles that are readily amenable to evaluation of compound pharmacology. Protocols are provided in this unit to obtain DMR response profiles in adherent and suspension cells, and then to use known tool compounds to delineate the biology of the underlying signaling pathways from the information-rich kinetic traces that are recorded. PMID:24652622

Gitschier, Hannah J; Bergeron, Audrey B; Randle, David H

2014-01-01

147

Progress in cell based assays for botulinum neurotoxin detection.  

PubMed

Botulinum neurotoxins (BoNTs) are the most potent human toxins known and the causative agent of botulism, and are widely used as valuable pharmaceuticals. The BoNTs are modular proteins consisting of a heavy chain and a light chain linked by a disulfide bond. Intoxication of neuronal cells by BoNTs is a multi-step process including specific cell binding, endocytosis, conformational change in the endosome, translocation of the enzymatic light chain into the cells cytosol, and SNARE target cleavage. The quantitative and reliable potency determination of fully functional BoNTs produced as active pharmaceutical ingredient (API) requires an assay that considers all steps in the intoxication pathway. The in vivo mouse bioassay has for years been the 'gold standard' assay used for this purpose, but it requires the use of large numbers of mice and thus causes associated costs and ethical concerns. Cell-based assays are currently the only in vitro alternative that detect fully functional BoNTs in a single assay and have been utilized for years for research purposes. Within the last 5 years, several cell-based BoNT detection assays have been developed that are able to quantitatively determine BoNT potency with similar or greater sensitivity than the mouse bioassay. These assays now offer an alternative method for BoNT potency determination. Such quantitative and reliable BoNT potency determination is a crucial step in basic research, in the development of pharmaceutical BoNTs, and in the quantitative detection of neutralizing antibodies. PMID:23239357

Pellett, Sabine

2013-01-01

148

Epitheliomesenchymal Transdifferentiation of Cultured RPE Cells  

Microsoft Academic Search

Retinal pigment epithelium (RPE) cells of the proliferative vitreoretinopathy (PVR) membrane take on the shape of fibroblasts and participate in fibrosis, thus deviating from the character of epithelial cells. This study was undertaken to evaluate RPE cell transdifferentiation in vitro. During the culture of porcine RPE cells, primary and 10th-passaged RPE cells were investigated for cell growth in response to

Sung-Chul Lee; Oh-Woong Kwon; Gong-Je Seong; Soon-Hyun Kim; Jae-Eun Ahn; Eun-Duck P. Kay

2001-01-01

149

Controlled, Scalable Embryonic Stem Cell Differentiation Culture  

Microsoft Academic Search

Embryonic stem (ES) cells are of significant interest as a renewable source of therapeutically useful cells. ES cell aggregation is important for both human and mouse embryoid body (EB) formation and the subse- quent generation of ES cell derivatives. Aggregation between EBs (agglomeration), however, inhibits cell growth and differentiation in stirred or high-cell-den- sity static cultures. We demonstrate that the

STEPHEN M. DANG; SHARON GERECHT-NIR; JINNY CHEN; JOSEPH ITSKOVITZ-ELDOR; PETER W. ZANDSTRAa

2004-01-01

150

Three-dimensional perfused cell culture.  

PubMed

Compelling evidence suggests the limitation and shortcomings of the current and well established cell culture method using multi-well plates, flasks and Petri dishes. These are particularly important when cell functions are sensitive to the local microenvironment, cell-cell and cell-extracellular matrix interactions. There is a clear need for advanced cell culture systems which mimic in vivo and more physiological conditions. This review summarises and analyses recent progress in three dimensional (3D) cell culture with perfusion as the next generation cell culture tools, while excluding engineered tissue culture where three dimensional scaffold has to be used for structural support and perfusion for overcoming mass transfer control. Apart from research activities in academic community, product development in industry is also included in this review. PMID:24184152

Li, Zhaohui; Cui, Zhanfeng

2014-01-01

151

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

152

Cell culture from sponges: pluripotency and immortality  

Microsoft Academic Search

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

Caralt Bosch de S; María J. Uriz; René H. Wijffels

2007-01-01

153

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...

154

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

155

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

156

Characterization of cultured multipotent zebrafish neural crest cells.  

PubMed

The neural crest is a unique cell population associated with vertebrate evolution. Neural crest cells (NCCs) are characterized by their multipotent and migratory potentials. While zebrafish is a powerful genetic model organism, the isolation and culture of zebrafish NCCs would provide a useful adjunct to fully interrogate the genetic networks that regulate NCC development. Here we report for the first time the isolation, in vitro culture, and characterization of NCCs from zebrafish embryos. NCCs were isolated from transgenic sox10:egfp embryos using fluorescence activated cell sorting and cultured in complex culture medium without feeder layers. NCC multilineage differentiation was determined by immunocytochemistry and real-time qPCR, cell migration was assessed by wound healing assay, and the proliferation index was calculated by immunostaining against the mitosis marker phospho-histone H3. Cultured NCCs expressed major neural crest lineage markers such as sox10, sox9a, hnk1, p75, dlx2a, and pax3, and the pluripotency markers c-myc and klf4. We showed that the cultured NCCs can be differentiated into multiple neural crest lineages, contributing to neurons, glial cells, smooth muscle cells, melanocytes, and chondrocytes. We applied the NCC in vitro model to study the effect of retinoic acid on NCC development. We showed that retinoic acid had a profound effect on NCC morphology and differentiation, significantly inhibited proliferation and enhanced cell migration. The availability of high numbers of NCCs and reproducible functional assays offers new opportunities for mechanistic studies of neural crest development, in genetic and chemical biology applications. PMID:24326414

Kinikoglu, Beste; Kong, Yawei; Liao, Eric C

2014-02-01

157

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting  

PubMed Central

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres. To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 °C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR. The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR. PMID:23051935

Venugopal, Chitra; McFarlane, Nicole M.; Nolte, Sara; Manoranjan, Branavan; Singh, Sheila K.

2012-01-01

158

[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

159

DNA repair: a simple enzymatic assay for human cells  

Microsoft Academic Search

An assay is described based on the ability of certain endonucleases in ; Micrococcus luteus extracts to react specifically at sites of DNA damage, and ; produce single-strand breaks. The breaks were detected by sedimenting the DNA ; through alkali. DNA repair was assumed to occur if the number of endonuclease ; sensitive sites decreased during post-treatment incubation of cells.

R. J. Wilkins

1973-01-01

160

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

161

MAMMALIAN CELL GENE MUTATION ASSAYS WORKING GROUP REPORT  

EPA Science Inventory

Mammalian cell gene mutation assays have been used for many years and the diversity of the available systems attests to the varied methods found to grow mammalian dells and detect mutations. s part of the International Workshop on Standardization of Genotoxicity Test Procedures, ...

162

A hybrid microfluidic platform for cell-based assays via diffusive and convective trans-membrane perfusion  

PubMed Central

We present a novel 3D hybrid assembly of a polymer microfluidic chip with polycarbonate track-etched membrane (PCTEM) enabling membrane-supported cell culture. Two chip designs have been developed to establish either diffusive or convective reagent delivery using the integrated PCTEM. While it is well suited to a range of cell-based assays, we specifically employ this platform for the screening of a common antitumor chemotoxic agent (mitomycin C – MMC) on the HL60 myeloid leukemia cell line. The toxic activity of MMC is based on the generation of severe DNA damage in the cells. Using either mode of operation, the HL60 cells were cultured on-chip before, during, and after exposure to MMC at concentrations ranging from 0 to 50??M. Cell viability was analysed off-chip by the trypan blue dye exclusion assay. The results of the on-chip viability assay were found to be consistent with those obtained off-chip and indicated ca. 40% cell survival at MMC concentration of 50??M. The catalogue of capabilities of the here described cell assay platform comprises of (i) the culturing of cells either under shear-free conditions or under induced through-membrane flows, (ii) the tight time control of the reagent exposure, (iii) the straightforward assembly of devices, (iv) the flexibility on the choice of the membrane, and, prospectively, (v) the amenability for large-scale parallelization. PMID:24404021

Vereshchagina, Elizaveta; Mc Glade, Declan; Glynn, Macdara; Ducree, Jens

2013-01-01

163

A comparative evaluation of various invasion assays testing colon carcinoma cell lines  

PubMed Central

Various colon carcinoma cell lines were tested in different invasion assays, i.e. invasion into Matrigel, into confluent fibroblast layers and into chicken heart tissue. Furthermore, invasive capacity and metastatic potential were determined in nude mice. The colon carcinoma cells used were the human cell lines Caco-2, SW-480, SW-620 and HT-29, and the murine lines Colon-26 and -38. None of the human colon carcinoma cells migrated through porous membranes coated with Matrigel; of the murine lines, only Colon-26 did. When incubated in a mixture of Matrigel and culture medium non-invading cells formed spheroid cultures, whereas invading cells showed a stellate outgrowth. Only the heterogeneously shaped (epithelioid and stellate) cells of SW-480 and SW-620 and the spindle-shaped cells of Colon-26 invaded clearly confluent skin and colon fibroblasts as well as chicken heart tissue. However, when transplanted into the caecum of nude and syngeneic mice, all the lines tested were invasive with the exception of Caco-2 cells. We conclude that the outcome of in vitro tests measuring the invasive capacity of neoplastic cells is largely dependent on the test system used. Invasive capacity in vitro is strongly correlated with cells having a spindle cell shape, vimentin expression and E-cadherin down regulation. In contrast, HT-29 and Colon-38 cells having an epithelioid phenotype were clearly invasive and metastatic in vivo, but not in vitro. © 1999 Cancer Research Campaign PMID:10576648

Both, N J de; Vermey, M; Dinjens, W N; Bosman, F T

1999-01-01

164

Pure cultures and characterization of yak Sertoli cells.  

PubMed

The culture of primary Sertoli cells has become an important resource in the study of their function. However, their use is limited because of contamination of isolated cells with other testicular cells, mainly germ cells. The aim was to establish technique to obtain pure yak Sertoli cells as well as to study the growth kinetics and biological characteristics of Sertoli cells in vitro. Two-step enzyme digestion was used to separate and culture yak Sertoli cells. Cultured using starvation method and the hypotonic treatment were also invented to get pure yak Sertoli cells. Furthermore, the purification of Yak Sertoli cells were identified according to their characteristics, such as bipolar corpuscular around the nucleus and expression of Fasl, in addition to their morphology. The average viability of the Sertoli cells was 97% before freezing and 94.5% after thawing, indicating that cryopreservation in liquid nitrogen had little influence on the viability of Sertoli cells. The growth tendency of yak Sertoli cells was similar to an S-shaped growth curve. Purified yak Sertoli cells frequently exhibited bipolar corpuscula in nucleus after Feulgen staining, and did have a positive reaction of Fasl by the immunocytochemical identification. After recovery chromosomal analysis of Sertoli cells had a normal chromosomal number of 60, comprising 29 pairs of autosomes and one pair of sex chromosomes. Assays for bacteria, fungi and mycoplasmas were negative. In conclusion, yak Sertoli cells have been successfully purified and cultured in vitro, and maintain stable biological characteristics after thawing. Therefore, it will not only preserve the genetic resources of yaks at the cellular level, but also provide valuable materials for transgenic research and feeder layer and nuclear donor cells in yak somatic cell cloning technology. PMID:23938058

Zhang, Hua; Liu, Ben; Qiu, Yuan; Fan, Jiang feng; Yu, Si jiu

2013-12-01

165

Single cell network profiling assay in bladder cancer.  

PubMed

The aim of this study was to assess the feasibility of applying the single cell network profiling (SCNP) assay to the examination of signaling networks in epithelial cancer cells, using bladder washings from 29 bladder cancer (BC) and 15 nonbladder cancer (NC) subjects. This report describes the methods we developed to detect rare epithelial cells (within the cells we collected from bladder washings), distinguish cancer cells from normal epithelial cells, and reproducibly quantify signaling within these low frequency cancer cells. Specifically, antibodies against CD45, cytokeratin, EpCAM, and cleaved-PARP (cPARP) were used to differentiate nonapoptotic epithelial cells from leukocytes, while measurements of DNA content to determine aneuploidy (DAPI stain) allowed for distinction between tumor and normal epithelial cells. Signaling activity in the PI3K and MAPK pathways was assessed by measuring intracellular levels of p-AKT and p-ERK at baseline and in response to pathway modulation; 66% (N = 19) of BC samples and 27% (N = 4) of NC samples met the "evaluable" criteria, i.e., at least 400,000 total cells available upon sample receipt with >2% of cells showing an epithelial phenotype. The majority of epithelial cells detected in BC samples were nonapoptotic and all signaling data were generated from identified cPARP negative cells. In four of 19 BC samples but in none of the NC specimens, SCNP assay identified epithelial cancer cells with a quantifiable increase in epidermal growth factor-induced p-AKT and p-ERK levels. Furthermore, preincubation with the PI3K inhibitor GDC-0941 reduced or completely inhibited basal and epidermal growth factor-induced p-AKT but, as expected, had no effect on p-ERK levels. This study demonstrates the feasibility of applying SCNP assay using multiparametric flow cytometry to the functional characterization of rare, bladder cancer cells collected from bladder washing. Following assay standardization, this method could potentially serve as a tool for disease characterization and drug development in bladder cancer and other solid tumors. PMID:23300058

Covey, Todd M; Vira, Manish A; Westfall, Matt; Gulrajani, Michael; Cholankeril, Michelle; Okhunov, Zhamshid; Levey, Helen R; Marimpietri, Carol; Hawtin, Rachael; Fields, Scott Z; Cesano, Alessandra

2013-04-01

166

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

167

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

168

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

169

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

170

Diagnostic performance and application of two commercial cell viability assays in foot-and-mouth disease research.  

PubMed

Cell-based assays are still used widely in foot-and-mouth disease (FMD) research, despite the existence of a wide variety of molecular techniques. The aim of this study was to validate an automated, quantitative spectrometric reading to replace the time-consuming and subjective microscopic (MIC) evaluation of the FMD virus-induced cytopathic effect (CPE). Therefore, the diagnostic performance of two commercial cell viability assays (CellTiter 96(®) AQueous One Solution Cell Proliferation Assay (MTS) and CellTiter-Blue(®) Cell Viability Assay (CTB), both from Promega, Leiden, The Netherlands) was evaluated. Following optimization of the assay protocols and using the MIC results as a reference standard, the absorbance-read MTS assay, the fluorescence-read CTB assay and the absorbance-read CTB (CTB(abs)) assay demonstrated similar high sensitivities (97%, 99% and 98%, respectively), specificities (100%, 98% and 99%, respectively), accuracy measures (0.99, 0.98 and 0.98, respectively), precision measures (1.00, 0.98 and 0.99, respectively) and Cohen kappa agreement indices (0.97, 0.97 and 0.96, respectively) for detecting CPE in cell cultures. Due to its performance, cost effectiveness and ease of use, the CTB(abs) assay was selected for further evaluation of its ability to detect virus neutralization and to screen antiviral compounds. The CTB(abs) assay had 99% sensitivity and 100% specificity for the detection of neutralizing antibodies in sera from cattle infected with FMDV and in sera from unvaccinated, uninfected cattle and resulted in a mean Z'-factor of 0.85 for antiviral compound test plates. The CTB(abs) assay is now used routinely in the Belgian FMD reference laboratory for serological testing and high-throughput antiviral compound screening. PMID:21295609

Willems, Tom; Lefebvre, David J; Neyts, Johan; De Clercq, Kris

2011-04-01

171

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

172

Glucosylation of taxifolin with cultured plant cells.  

PubMed

Cultured plant cells of Eucalyptus perriniana glucosylated taxifolin to its 3'- and 7-O-beta-D-glucosides and 3',7-O-beta-D-diglucoside. On the other hand, taxifolin was converted into 3'- and 7-O-beta-D-glucosides by cultured cells of Nicotiana tabacum and Catharanthus roseus. PMID:23980419

Shimoda, Kei; Kubota, Naoji; Hamada, Manabu; Sugamoto, Masahiro; Ishihara, Kohji; Hamada, Hatsuyuki; Hamada, Hiroki

2013-07-01

173

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...

174

Marine Invertebrate Cell Cultures: New Millennium Trends  

Microsoft Academic Search

This review analyzes activities in the field of marine invertebrate cell culture during the years 1999 to 2004 and compares the outcomes with those of the preceding decade (1988 to 1998). During the last 5 years, 90 reports of primary cell culture studies of marine organisms belonging to only 6 taxa (Porifera, Cnidaria, Crustacea, Mollusca, Echinodermata, and Urochordata) have been

Baruch Rinkevich

2005-01-01

175

Pokeweed mitogen induced differentiation of human B cells: evaluation by a protein A haemolytic plaque assay.  

PubMed Central

Using the protein A plaque assay, the number of human cells secreting immunoglobulin of various classes after pokeweed mitogen stimulation was determined. At optimal response (on day 5-7) a mean of 58,354 IgM PFC/10(6), 34,207 IgG PFC/10(6) and 10,525 IgA PFC/10(6) cells was found when using peripheral blood lymphocytes. In spleen cells, peak values which were slightly higher than in blood, were obtained at day 4-6. The proportions of cells secreting light chains of either type were found to be comparable to those of unstimulated cells thus supporting the notion of the polyclonality of the response. Pokeweed mitogen stimulation of peripheral blood lymphocytes was found to be totally T-cell dependent whereas the response of spleen cells was not. When assayed for antigen-specific precursor cells in cultures stimulated by mitogen, the frequency of SRBC-specific IgM producing cells was found to be 1.3/1000 cells. This frequency was regularly found to be independent of medium supplement. PMID:116958

Hammarstrom, L; Bird, A G; Britton, S; Smith, C I

1979-01-01

176

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

177

Evaluation of Tualang honey as a supplement to fetal bovine serum in cell culture  

Microsoft Academic Search

The aim of this study was to evaluate Tualang honey as a supplement to fetal bovine serum in cell cultures using MTT assay, chromosome aberration test and gene expression analyses. The MTT assay showed the highest percentage of cell proliferation (105.3% increment than control) of human osteoblast cell line (CRL 1543) in 0.0195% honey in Dulbecco’s modified eagle medium supplemented

Thirumulu Ponnuraj Kannan; Abdulaziz Qaid Ali; Siti Fadilah Abdullah; Azlina Ahmad

2009-01-01

178

Mammalian cell cultures for biologics manufacturing.  

PubMed

Biopharmaceuticals represent a growing sector of the pharmaceutical industry, and are used for a wide range of indications, including oncology and rheumatology. Cultured mammalian cells have become the predominant expression system for their production, partly due to their ability to complete the posttranslational modifications required for drug safety and efficacy. Over the past decade, the productivity of mammalian cell culture production processes has growth dramatically through improvements in both volumetric and specific productivities. This article presents an overview of the biologics market, including analysis of sales and approvals; as well as a review of industrial production cell lines and cell culture operations. PMID:24258145

Kantardjieff, Anne; Zhou, Weichang

2014-01-01

179

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

180

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

181

Cell assay using a two-photon-excited europium chelate  

PubMed Central

We report application of two-photon excitation of europium chelates to immunolabeling of epidermal growth factor receptor (EGFR) cell surface proteins on A431 cancer cells. The europium chelates are excited with two photons of infrared light and emit in the visible. Europium chelates are conjugated to antibodies for EGFR. A431 (human epidermoid carcinoma) cells are labeled with this conjugate and imaged using a multiphoton microscope. To minimize signal loss due to the relatively long-lived Eu3+ emission, the multiphoton microscope is used with scanning laser two-photon excitation and non-scanning detection with a CCD. The chelate labels show very little photobleaching (less than 1% during continuous illumination in the microscope for 20 minutes) and low levels of autofluorescence (less than 1% of the signal from labeled cells). The detection limit of the europium label in the cell assay is better than 100 zeptomoles. PMID:21833362

Xiao, Xudong; Haushalter, Jeanne P.; Kotz, Kenneth T.; Faris, Gregory W.

2011-01-01

182

High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays.  

PubMed

Heterogeneity in cell populations poses a major obstacle to understanding complex biological processes. Here we present a microfluidic platform containing thousands of nanoliter-scale chambers suitable for live-cell imaging studies of clonal cultures of nonadherent cells with precise control of the conditions, capabilities for in situ immunostaining and recovery of viable cells. We show that this platform mimics conventional cultures in reproducing the responses of various types of primitive mouse hematopoietic cells with retention of their functional properties, as demonstrated by subsequent in vitro and in vivo (transplantation) assays of recovered cells. The automated medium exchange of this system made it possible to define when Steel factor stimulation is first required by adult hematopoietic stem cells in vitro as the point of exit from quiescence. This technology will offer many new avenues to interrogate otherwise inaccessible mechanisms governing mammalian cell growth and fate decisions. PMID:21602799

Lecault, Véronique; Vaninsberghe, Michael; Sekulovic, Sanja; Knapp, David J H F; Wohrer, Stefan; Bowden, William; Viel, Francis; McLaughlin, Thomas; Jarandehei, Asefeh; Miller, Michelle; Falconnet, Didier; White, Adam K; Kent, David G; Copley, Michael R; Taghipour, Fariborz; Eaves, Connie J; Humphries, R Keith; Piret, James M; Hansen, Carl L

2011-07-01

183

Oxygen Control in Static Cell Cultures  

Microsoft Academic Search

Oxygen control in static cell cultures is one of the most critical parameters of a process, influencing the metabolism of\\u000a the cells and ultimately the final yield of the product. With the aim to improve the cultures of different types of cells,\\u000a both anchorage dependent and suspension, we have carried out several experiments evaluating the behaviour of some types of

Nadia De Bernardi; Edwin Schwander; Antonio Orlandi; Ilaria Tano; Sonia Castiglioni; Elena Muru; Fausto Gaspari; Marta Galgano; Claudia Mattei; Luigi Cavenaghi; Maria Nolli

184

Recommended protocol for the BALB/c 3T3 cell transformation assay.  

PubMed

The present protocol has been developed for the BALB/c 3T3 cell transformation assay (CTA), following the prevalidation study coordinated by the European Centre for the Validation of Alternative Methods (ECVAM) and reported in this issue (Tanaka et al. [16]). Based upon the experience gained from this effort and as suggested by the Validation Management Team (VMT), some acceptance and assessment criteria have been refined compared to those used during the prevalidation study. The present protocol thus describes cell culture maintenance, the dose-range finding (DRF) experiment and the transformation assay, including cytotoxicity and morphological transformation evaluation. Use of this protocol and of the associated photo catalogue included in this issue (Sasaki et al. [17]) is recommended for the future conduct of the BALB/c 3T3 CTA. PMID:22212201

Sasaki, Kiyoshi; Bohnenberger, Susanne; Hayashi, Kumiko; Kunkelmann, Thorsten; Muramatsu, Dai; Phrakonkham, Pascal; Poth, Albrecht; Sakai, Ayako; Salovaara, Susan; Tanaka, Noriho; Thomas, B Claire; Umeda, Makoto

2012-04-11

185

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

186

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

PubMed

Current therapy for pancreatic cancer is multimodal, involving surgery and chemotherapy. However, development of pancreatic cancer therapies requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Compared to two-dimensional culture of cell monolayer, three-dimensional (3-D) models more closely mimic native tissues, since the tumor microenvironment established in 3-D models often plays a significant role in cancer progression and cellular responses to the drugs. Accumulating evidence has highlighted the benefits of 3-D in vitro models of various cancers. In the present study, we have developed a spheroid-based, 3-D culture of pancreatic cancer cell lines MIAPaCa-2 and PANC-1 for pancreatic drug testing, using the acid phosphatase assay. Drug efficacy testing showed that spheroids had much higher drug resistance than monolayers. This model, which is characteristically reproducible and easy and offers rapid handling, is the preferred choice for filling the gap between monolayer cell cultures and in vivo models in the process of drug development and testing for pancreatic cancer. PMID:23903680

Wen, Z; Liao, Q; Hu, Y; You, L; Zhou, L; Zhao, Y

2013-07-01

187

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

188

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

189

A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers.  

PubMed

A method to simultaneously determine the relative numbers of live and dead cells in culture by introducing a combination of two fluorogenic substrates or a fluorogenic and a luminogenic protease substrate into the sample is described. The method is based on detection of differential ubiquitous proteolytic activities associated with intact viable cells and cells that have lost membrane integrity. A cell-permeable peptide aminofluorocoumarin substrate detects protease activity restricted to intact viable cells. Upon cell death, the viable cell protease marker becomes inactive. An impermeable peptide rhodamine 110 (or aminoluciferin) conjugated substrate detects protease activity from nonviable cells that have lost membrane integrity. The multiplex assay can detect 200 dead cells in a population of 10,000 viable cells. The protease substrate reagents do not damage viable cells over the course of the assay, thus the method can be multiplexed further with other assays in a homogeneous format. Ratiometric measurement of viable and dead cells in the same sample provides an internal control that can be used to normalize data from other cell-based assays. PMID:17512890

Niles, Andrew L; Moravec, Richard A; Eric Hesselberth, P; Scurria, Michael A; Daily, William J; Riss, Terry L

2007-07-15

190

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

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

Draining lymph node cell activation in guinea pigs: comparisons with the murine local lymph node assay.  

PubMed

The local lymph node assay in the mouse is a novel predictive test for the identification of contact sensitizing chemicals. The purpose of the studies described was to determine whether a similar local lymph node assay could be performed successfully in guinea pigs; currently the species of choice for assessment of sensitizing potential for regulatory purposes. Ten sensitizing chemicals (oxazolone, picryl chloride, 2,4-dinitrofluorobenzene, benzocaine, cinnamic aldehyde, 2,4,-dinitrothiocyanobenzene, p-nitrosodimethylaniline, formaldehyde, p-phenylenediamine and cyanuric chloride) and equal concentrations of sodium lauryl sulphate were examined in a guinea pig local lymph node assay. Animals received three consecutive daily applications of various concentrations of the test chemical on the dorsum of both ears. Control animals were untreated. Five days following the initiation of exposure, draining auricular lymph nodes were excised and weighed. Suspensions of lymph node cells (LNC) were prepared and cultured for 24 or 48 h and proliferation measured by incorporation of [3H]thymidine. Exposure to at least one concentration of all sensitizing chemicals, other than benzocaine, induced proliferation by draining LNC. Responses were higher at 24 h rather than 48 h. Evidence is presented that guinea pig LNC proliferation may be enhanced or maintained by addition to culture of an exogenous source of the T cell growth factor interleukin 2 (IL-2). Draining lymph node weight was increased following exposure to some sensitizing chemicals but, compared with LNC proliferation, provided a less sensitive correlate of lymph node activation. Exposure to sodium lauryl sulphate failed to induce changes in either lymph node weight of LNC proliferation. Data are compared with three-day murine local lymph node assays performed concurrently. The available information indicates that the local lymph node assay may be performed in guinea pigs. PMID:1949049

Maurer, T; Kimber, I

1991-01-01

193

Adipose cell differentiation in culture  

Microsoft Academic Search

The isolation of preadipocyte cell strains from adipose tissue and from bone marrow, and the establishment of preadipocyte cell lines from embryonic and adult mouse, have been useful tools to study the process of adipose cell differentiation.

G. Ailhaud

1982-01-01

194

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model  

PubMed Central

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS. The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13. We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS. PMID:22297395

Machado, Heather L.; Morris, Cindy A.; Höner zu Bentrup, Kerstin

2012-01-01

195

A microfluidic localized, multiple cell culture array using vacuum actuated cell seeding: integrated anticancer drug testing.  

PubMed

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 cell 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; Pappas, Dimitri

2013-12-01

196

Development of culture-based serological assays to diagnose Babesia divergens infections.  

PubMed

Babesioses are hematic tick-borne diseases that induce malaria-like disorders in domestic, wild animals, and humans. Although indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) commercial kits are available to test the presence of antibodies against most Babesia species, no kit exists to serologically diagnose the infections due to Babesia divergens, one of the most important zoonotic species. To fill this gap and to develop assays to detect animal and human infections, in vitro cultures (microaerophilous stationary phase system) of B. divergens were organized. Infected erythrocytes were adsorbed as corpuscular antigen (CA) on IFAT slides and ELISA microwells. The supernatant medium of the cultures (metabolic antigen, MA) was collected and employed in ELISA and western blot (WB) assays. B. divergens was also used to produce positive sera in Meriones unguiculatus and to infect a calf. Serological tests were set up with sera from experimentally/naturally infected animals, and possible cross-reactions were evaluated using heterologous sera from cattle positive to other piroplasms. Sera from clinically healthy people at risk of infection were also tested. As expected, assays based on the purified MAs from in vitro cultures proved more sensitive and specific than CA-IFAT and CA-ELISA. In fact, MA-ELISA provided satisfactory performances (even if 8.4%-15.7% cross-reactions were evidenced), and the WB developed proved totally sensitive and specific. WB indicated as immunodominant antigens two major protein bands at 33 and 37?kDa, which were also evidenced in 2.2% of the human sera tested, proving the parasite transmission to humans also in Italy. PMID:21995263

Gabrielli, Simona; Galuppi, Roberta; Marcer, Federica; Marini, Carla; Tampieri, Maria Paola; Moretti, Annabella; Pietrobelli, Mario; Cancrini, Gabriella

2012-02-01

197

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

198

Cell transformation assays: are we barking up the wrong tree?  

PubMed

There has been a current resurgence of interest in the use of cell transformation for predicting carcinogenicity, which is based mainly on rodent carcinogenicity data. In view of this renewed interest, this paper critically reviews the published literature concerning the ability of the available assays to detect IARC Group 1 agents (known human carcinogens) and Group 2A agents (probable human carcinogens). The predictivity of the available assays for human and rodent non-genotoxic carcinogens (NGCs), in comparison with standard and supplementary in vitro and in vivo genotoxicity tests, is also discussed. The principal finding is that a surprising number of human carcinogens have not been tested for cell transformation across the three main assays (SHE, Balb/c 3T3 and C3H10T1/2), confounding comparative assessment of these methods for detecting human carcinogens. This issue is not being addressed in the ongoing validation studies for the first two of these assays, despite the lack of any serious logistical issues associated with the use of most of these chemicals. In addition, there seem to be no plans for using exogenous bio-transformation systems for the metabolic activation of pro-carcinogens, as recommended in an ECVAM workshop held in 1999. To address these important issues, it is strongly recommended that consideration be given to the inclusion of more human carcinogens and an exogenous source of xenobiotic metabolism, such as an S9 fraction, in ongoing and future validation studies. While cell transformation systems detect a high level of NGCs, it is considered premature to rely only on this endpoint for screening for such chemicals, as recently suggested. This is particularly important, in view of the fact that there is still doubt as to the relevance of morphological transformation to tumorigenesis in vivo, and the wide diversity of potential mechanisms by which NGCs are known to act. Recent progress with regard to increasing the objectivity of scoring the transformed phenotype, and prospects for developing human cell-based transformation assays, are reviewed. PMID:22762196

Combes, Robert D

2012-05-01

199

Bioprocessing technology for plant cell suspension cultures  

Microsoft Academic Search

Considering various forms of in vitro plant tissue cultures, cell suspension culture is most amenable to large-scale production\\u000a of natural compounds, owing primarily to its superior culture homogeneity. This fact has already been demonstrated in several\\u000a largescale applications, including the commercial shikonin process. The scope of this work is to review the state of the art\\u000a in bioprocessing technologies pertinent

Wei wen Su

1995-01-01

200

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

201

Culture and manipulation of embryonic cells.  

PubMed

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

202

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(9) 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

2014-01-01

203

Verbascoside production by plant cell cultures  

Microsoft Academic Search

Verbascoside was found to be produced in all calli derived from eleven species that contained the compound in their leaves. Cell suspension cultures were also established in three species, i.e., Leucosceptrum japonicum f. barbinerve, Syringa josikaea, and Sy. vulgaris, all of which were found to produce verbascoside at more than 1 g\\/l. Of the three species, suspension cultures of L.

Nobuyuki Inagaki; Hiroaki Nishimura; Minoru Okada; Hiroshi Mitsuhashi

1991-01-01

204

Haute Culture: Tailoring stem cells  

E-print Network

Biology, Department of Stem Cell and Regenerative Biology, Harvard University Massachusetts General Hospital Fernando Camargo, PhD Assistant Professor of Stem Cell Regenerative Biology, Department of Stem Cell and Regenerative Biology, Harvard University Children's Hospital Boston Stem Cell Program #12

Chou, James

205

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

206

Rapid Identification of Burkholderia pseudomallei in Blood Cultures by a Monoclonal Antibody Assay  

PubMed Central

Burkholderia pseudomallei is the causative agent of melioidosis. In northeast Thailand, this gram-negative bacterium is a major cause of mortality from septicemia. The definitive diagnosis of this disease is made by bacterial culture. In this study, we produced a monoclonal antibody (MAb) specific to the 30-kDa protein of B. pseudomallei by in vivo and in vitro immunization of BALB/c mice with a crude culture filtrate antigen. The MAb could directly agglutinate with all 243 clinical isolates of B. pseudomallei but not with other gram-negative bacteria, except for one strain of Burkholderia mallei. However, the MAb cross-reacted with the gram-positive Bacillus sp. and Streptococcus pyogenes. B. pseudomallei in brain heart infusion broth (BHIB) subcultured from a BacT/Alert automated blood culture system could be identified by simple agglutination with this MAb assay. The sensitivity and specificity of direct agglutination compared to the “gold standard,” the culture method, were 94.12 and 98.25%, respectively. However, the MAb adsorbed to polystyrene beads or latex particles directly identified the bacterium in blood culture specimens and in BHIB subcultured from a BacT/Alert automated blood culture system. The sensitivity of the latex agglutination test was 100% for both blood culture and BHIB specimens. The specificity was 85.96 and 96.49% for the blood culture and BHIB specimens, respectively. The specificity could be increased if the nonspecific materials in the blood culture broths were eradicated by centrifugation at low speeds. Thus, a combination of blood culture and the agglutination method could be used for the rapid diagnosis of melioidosis in the routine bacteriological laboratory. This method could speed up detection of the bacterium in blood culture by at least 2 days, compared to the conventional bacterial culture method. In addition, the MAb is stable at room temperature for 2 weeks and at 4, ?20, and ?70°C for at least 1 year. The latex reagent was stable for at least 6 months at 4°C. PMID:10523570

Pongsunk, Supinya; Thirawattanasuk, Nittaya; Piyasangthong, Nuanchan; Ekpo, Pattama

1999-01-01

207

Cell culture systems for hepatitis C virus.  

PubMed

Due to the obligatory intracellular lifestyle of viruses, cell culture systems for efficient viral propagation are crucial to obtain a detailed understanding of the virus-host cell interaction. For hepatitis C virus (HCV) the development of permissive and authentic culture models continues to be a challenging task. The first efforts to culture HCV had limited success and range back to before the virus was molecularly cloned in 1989. Since then several major breakthroughs have gradually overcome limitations in culturing the virus and sequentially permitted analysis of viral RNA replication, cell entry, and ultimately the complete replication cycle in cultured cells in 2005. Until today, basic and applied HCV research greatly benefit from these tremendous efforts which spurred multiple complementary cell-based model systems for distinct steps of the HCV replication cycle. When used in combination they now permit deep insights into the fascinating biology of HCV and its interplay with the host cell. In fact, drug development has been much facilitated and our understanding of the molecular determinants of HCV replication has grown in parallel to these advances. Building on this groundwork and further refining our cellular models to better mimic the architecture, polarization and differentiation of natural hepatocytes should reveal novel unique aspects of HCV replication. Ultimately, models to culture primary HCV isolates across all genotypes may teach us important new lessons about viral functional adaptations that have evolved in exchange with its human host and that may explain the variable natural course of hepatitis C. PMID:23463196

Steinmann, Eike; Pietschmann, Thomas

2013-01-01

208

Direct 5S rRNA Assay for Monitoring Mixed-Culture Bioprocesses  

PubMed Central

This study demonstrates the efficacy of a direct 5S rRNA assay for the characterization of mixed microbial populations by using as an example the bacteria associated with acidic mining environments. The direct 5S rRNA assay described herein represents a nonselective, direct molecular method for monitoring and characterizing the predominant, metabolically active members of a microbial population. The foundation of the assay is high-resolution denaturing gradient gel electrophoresis (DGGE), which is used to separate 5S rRNA species extracted from collected biomass. Separation is based on the unique migration behavior of each 5S rRNA species during electrophoresis in denaturing gradient gels. With mixtures of RNA extracted from laboratory cultures, the upper practical limit for detection in the current experimental system has been estimated to be greater than 15 different species. With this method, the resolution was demonstrated to be effective at least to the species level. The strength of this approach was demonstrated by the ability to discriminate between Thiobacillus ferrooxidans ATCC 19859 and Thiobacillus thiooxidans ATCC 8085, two very closely related species. Migration patterns for the 5S rRNA from members of the genus Thiobacillus were readily distinguishable from those of the genera Acidiphilium and Leptospirillum. In conclusion, the 5S rRNA assay represents a powerful method by which the structure of a microbial population within acidic environments can be assessed. PMID:16535333

Stoner, D. L.; Browning, C. K.; Bulmer, D. K.; Ward, T. E.; MacDonell, M. T.

1996-01-01

209

Establishment and Characterization of a Madin-Darby Canine Kidney Reporter Cell Line for Influenza A Virus Assays?  

PubMed Central

Influenza virus diagnosis has traditionally relied on virus isolation in chicken embryo or cell cultures. Many laboratories have adopted rapid molecular methods for detection of influenza viruses and discontinued routine utilization of the relatively slow viral culture methods. We describe an influenza A virus reporter cell line that contributes to more efficient viral detection in cell culture. Madin-Darby canine kidney (MDCK) cells were engineered to constitutively produce an influenza virus genome-like luciferase reporter RNA driven by the canine RNA polymerase I promoter. Induction of a high level of luciferase activity was detected in the Luc9.1 cells upon infection with various strains of influenza A virus, including 2009 H1N1 pandemic and highly pathogenic H5N1 virus. In contrast, infection with influenza B virus or human adenovirus type 5 did not induce significant levels of reporter expression. The reporter Luc9.1 cells were evaluated in neutralizing antibody assays with convalescent H3N2 ferret serum, yielding a neutralization titer comparable to that obtained by the conventional microneutralization assay, suggesting that the use of the reporter cell line might simplify neutralization assays by facilitating the establishment of infectious virus endpoints. Luc9.1 cells were also used to determine the susceptibility of influenza A viruses to a model antiviral drug. The equivalence to conventional antiviral assay results indicated that the Luc9.1 cells could provide an alternative cell-based platform for high-throughput drug discovery screens. In summary, the MDCK-derived Luc9.1 reporter cell line is highly permissive for influenza A virus replication and provides a very specific and sensitive approach for simultaneous detection and isolation of influenza A viruses as well as functional evaluation of antibodies and antiviral molecules. PMID:20504984

Hossain, M. Jaber; Perez, Sandra; Guo, Zhu; Chen, Li-Mei; Donis, Ruben O.

2010-01-01

210

Automated adherent human cell culture (mesenchymal stem cells).  

PubMed

Human cell culture processes developed at research laboratory scale need to be translated to large-scale production processes to achieve commercial application to a large market. To allow this transition of scale with consistent process performance and control of costs, it will be necessary to reduce manual processing and increase automation. There are a number of commercially available platforms that will reduce manual process intervention and improve process control for different culture formats. However, in many human cell-based applications, there is currently a need to remain close to the development format, usually adherent culture on cell culture plastic or matrix-coated wells or flasks due to deterioration of cell quality in other environments, such as suspension. This chapter presents an example method for adherent automated human stem cell culture using a specific automated flask handling platform, the CompacT SelecT. PMID:22057466

Thomas, Robert; Ratcliffe, Elizabeth

2012-01-01

211

Enrichment of tumor-initiating breast cancer cells within a mammosphere-culture microdevice.  

PubMed

We report for the first time a microdevice that enables the selective enrichment, culture, and identification of tumor-initiating cells on native polydimethylsiloxane (PDMS). For nearly a decade, researchers have identified tumor-initiating breast cancer cells within heterogeneous populations of breast cancer cells by utilizing low-attachment serum-free culture conditions, which lead to the formation of spheroidal colonies (mammospheres) that are enriched for tumor-initiating cells. However, the utility of this assay has been limited by difficulties in combining this culture-plate-based technique with other cellular and molecular analyses. Integrating the mammosphere technique into a microsystem can enable it to be combined directly with a number of functions, such as cell sorting, drug screens, and molecular assays. In this work, we demonstrate mammosphere culture within a PDMS microdevice. We first prove that a native hydrophobic PDMS surface is as effective as commercial low-attachment plates at selectively promoting the formation of mammospheres. We then experimentally assess the PDMS microdevice. Time-lapse images of mammosphere formation within the microdevice show that mammospheres form from single cells or small clusters of cells. Following formation of the mammospheres, it is desirable to evaluate the cells within the spheroids for enrichment of tumor initiating cells. To perform assays such as this (which require the loading and rinsing of reagents) without flushing the cells (which are in suspension) from the device, the culture chamber is separated from a reagent reservoir by a commercially available microporous membrane, and thus reagents are exchanged between the reservoir and the culture chamber by diffusion only. Using this capability, we verify that the mammospheres are enriched for tumor initiating cells by staining aldehyde dehydrogenase activity, a cancer stem cell marker. To the best of our knowledge, this is the first assay that enables the direct observation of tumor-initiating cells within a suspended mammosphere. PMID:23515914

Saadin, Katayoon; Burke, Jeffrey M; Patel, Neerav P; Zubajlo, Rebecca E; White, Ian M

2013-08-01

212

Bone Marrow Mononuclear Cells Protect Neurons and Modulate Microglia in Cell Culture Models of Ischemic Stroke  

PubMed Central

Background Although several studies have provided evidence for the therapeutic potential of bone marrow-derived mononuclear cells (MNCs) in animal models of stroke, the mechanisms underlying their benefits remain largely unknown. We have determined the neuroprotective potential of MNCs in primary neuronal cultures exposed to various injuries in vitro. Methods Cortical neurons in culture were exposed to oxygen-glucose deprivation, hypoxia, or hydrogen peroxide and cell death was assayed by MTT, caspase-3 activation or TUNEL labelling at 24 hrs. Cultures were randomized to co-treatment with MNC-derived supernatants or media before injury exposure. In separate experiments, macrophage or microglial cultures were exposed to lipopolypolysacharide (LPS) in the presence and absence of MNC-derived supernatants. Neuronal cultures were then exposed to conditioned media derived from activated macrophages or microglia. Cytokines from the supernantants of MNC cultures exposed to normoxia or hypoxia were also estimated by enzyme-linked immunosorbant assay (ELISA). Results MNC-derived supernatants attenuated neuronal death induced by OGD, hypoxia, hydrogen peroxide, and conditioned macrophage/microglial media and contain a number of trophic factors including IL-10, IGF-1, VEGF, and SDF-1. Conclusion MNCs provide broad neuroprotection against a variety of injuries relevant to stroke. PMID:20629187

Sharma, Sushil; Yang, Bing; Strong, Roger; Xi, Xiao Pei; Brenneman, Miranda; Grotta, James C.; Aronowski, Jaroslaw; Savitz, Sean I.

2010-01-01

213

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes  

PubMed Central

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells. PMID:19373242

Galluzzi, L; Aaronson, SA; Abrams, J; Alnemri, ES; Andrews, DW; Baehrecke, EH; Bazan, NG; Blagosklonny, MV; Blomgren, K; Borner, C; Bredesen, DE; Brenner, C; Castedo, M; Cidlowski, JA; Ciechanover, A; Cohen, GM; De Laurenzi, V; De Maria, R; Deshmukh, M; Dynlacht, BD; El-Deiry, WS; Flavell, RA; Fulda, S; Garrido, C; Golstein, P; Gougeon, M-L; Green, DR; Gronemeyer, H; Hajn?czky, G; Hardwick, JM; Hengartner, MO; Ichijo, H; Jaattela, M; Kepp, O; Kimchi, A; Klionsky, DJ; Knight, RA; Kornbluth, S; Kumar, S; Levine, B; Lipton, SA; Lugli, E; Madeo, F; Malorni, W; Marine, J-CW; Martin, SJ; Medema, JP; Mehlen, P; Melino, G; Moll, UM; Morselli, E; Nagata, S; Nicholson, DW; Nicotera, P; Nunez, G; Oren, M; Penninger, J; Pervaiz, S; Peter, ME; Piacentini, M; Prehn, JHM; Puthalakath, H; Rabinovich, GA; Rizzuto, R; Rodrigues, CMP; Rubinsztein, DC; Rudel, T; Scorrano, L; Simon, H-U; Steller, H; Tschopp, J; Tsujimoto, Y; Vandenabeele, P; Vitale, I; Vousden, KH; Youle, RJ; Yuan, J; Zhivotovsky, B; Kroemer, G

2009-01-01

214

Glycosylation of hesperetin by plant cell cultures  

Microsoft Academic Search

The biotransformation of hesperetin by cultured cells of Ipomoea batatas and Eucalyptus perriniana was investigated. Three glycosides, hesperetin 3?-O-?-d-glucopyranoside (33?g\\/g fr. wt of cells), hesperetin 3?,7-O-?-d-diglucopyranoside (217?g\\/g fr. wt of cells), and hesperetin 7-O-[6-O-(?-d-glucopyranosyl)]-?-d-glucopyranoside (?-gentiobioside, 22?g\\/g fr. wt of cells), together with three hitherto known glycosides, hesperetin 5-O-?-d-glucopyranoside (23?g\\/g fr. wt of cells), hesperetin 7-O-?-d-glucopyranoside (57?g\\/g fr. wt of cells),

Kei Shimoda; Hatsuyuki Hamada; Hiroki Hamada

2008-01-01

215

Isolation of Nuclei from Skeletal Muscle Satellite Cells and Myofibers for Use in Chromatin lmmunoprecipitation Assays  

PubMed Central

Studies investigating mechanisms controlling gene regulation frequently examine specific DNA sequences using chromatin immunoprecipitation (ChIP) assays to determine whether specific regulatory factors or modified histones are present. While use of primary cells or cell line models for differentiating or differentiated tissue is widespread, the ability to assess factor binding and histone modification in tissue defines the events that occur in vivo and provides corroboration for studies in cultured cells. Many tissues can be analyzed with minimal modification to existing ChIP protocols that are designed for cultured cells; however, some tissues, such as skeletal muscle, are problematic in that accessibility of the cross-linking agent is limited. We describe a method to isolate skeletal muscle tissue nuclei suitable for use in ChIP protocols. Furthermore, we utilize a simple fractionation of digested skeletal muscle tissue that can separate mature myofibers from satellite cells, which are responsible for postnatal skeletal muscle regeneration, thereby allowing simultaneous preparation of nuclei from both cell types. PMID:22130858

Ohkawa, Yasuyuki; Mallappa, Chandrashekara; Dacwag Vallaster, Caroline S.; lmbalzano, Anthony N.

2014-01-01

216

Henrietta Lacks, HeLa cells, and cell culture contamination.  

PubMed

Henrietta Lacks died in 1951 of an aggressive adenocarcinoma of the cervix. A tissue biopsy obtained for diagnostic evaluation yielded additional tissue for Dr George O. Gey's tissue culture laboratory at Johns Hopkins (Baltimore, Maryland). The cancer cells, now called HeLa cells, grew rapidly in cell culture and became the first human cell line. HeLa cells were used by researchers around the world. However, 20 years after Henrietta Lacks' death, mounting evidence suggested that HeLa cells contaminated and overgrew other cell lines. Cultures, supposedly of tissues such as breast cancer or mouse, proved to be HeLa cells. We describe the history behind the development of HeLa cells, including the first published description of Ms Lacks' autopsy, and the cell culture contamination that resulted. The debate over cell culture contamination began in the 1970s and was not harmonious. Ultimately, the problem was not resolved and it continues today. Finally, we discuss the philosophical implications of the immortal HeLa cell line. PMID:19722756

Lucey, Brendan P; Nelson-Rees, Walter A; Hutchins, Grover M

2009-09-01

217

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

218

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; Kluter, Harald; Bugert, Peter

2014-01-01

219

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

220

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

221

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

PubMed

No gold standard assay exhibiting error-free classification of results has been identified for detection of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Validation of diagnostic assays for R. salmoninarum has been hindered by its unique characteristics and biology, and difficulties in locating suitable populations of reference test animals. Infection status of fish in test populations is often unknown, and it is commonly assumed that the assay yielding the most positive results has the highest diagnostic accuracy, without consideration of misclassification of results. In this research, quantification of R. salmoninarum in samples by bacteriological culture provided a standardized measure of viable bacteria to evaluate analytical performance characteristics (sensitivity, specificity and repeatability) of non-culture assays in three matrices (phosphate-buffered saline, ovarian fluid and kidney tissue). Non-culture assays included polyclonal enzyme-linked immunosorbent assay (ELISA), direct smear fluorescent antibody technique (FAT), membrane-filtration FAT, nested polymerase chain reaction (nested PCR) and three real-time quantitative PCR assays. Injection challenge of specific pathogen-free Chinook salmon, Oncorhynchus tshawytscha (Walbaum), with R. salmoninarum was used to estimate diagnostic sensitivity and specificity. Results did not identify a single assay demonstrating the highest analytical and diagnostic performance characteristics, but revealed strengths and weaknesses of each test. PMID:23346868

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

2013-09-01

222

Rosmarinic acid production in Coleus cell cultures  

Microsoft Academic Search

Cell suspension cultures of Coleus blumei Benth. have been found to accumulate 8–11% of their dry weight as rosmarinic acid (a-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

A. Razzaque; B. E. Ellis

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

Estrogen receptor in dissociated and cultured human breast fibroadenoma epithelial cells.  

PubMed

Estrogen binding was measured by a whole cell receptor assay in epithelial cells isolated from 20 premenopausal patients with breast fibroadenomas. A high affinity specific binding for estrogens was detected in the epithelial cells isolated from all 20 fibroadenomas. A relationship between estrogen binding and the phase in the menstrual cycle of the patient has been observed. Cell culture experiments using serum-free medium have also shown that estrogen binding can be augmented by cortisol. PMID:3828977

Balakrishnan, A; Yang, J; Beattie, C W; Das Gupta, T K; Nandi, S

1987-03-01

225

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

226

Multiplex real-time PCR assay for rapid detection of methicillin-resistant staphylococci directly from positive blood cultures.  

PubMed

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 10(3) 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; Uh, Young; Lee, Hyeyoung

2014-06-01

227

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

228

Excessive stem cell competition in the competitive long-term repopulation assay - development of a solution  

Microsoft Academic Search

The competitive long-term repopulating stem cell assay is widely used as the standard method for detecting murine hematopoietic stem cells. An integral part of this assay is the competition between the test stem cells and those in the irradiated recipient, as well as the carrier cell population which ensures the short-term survival of the animal. Recently, we and others have

I. Ponting; H.-M. Wang; L. Chiu; A. Shpaner; F. Shin

2000-01-01

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

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

231

Probing Enzymatic Activity inside Living Cells Using a Nanowire-Cell "Sandwich" Assay  

E-print Network

of an intracellular signal transduction pathway requires minimally invasive methods for probing enzyme activity in situ. Here, we describe a new method for monitoring enzyme activity in living cells by sandwiching live cell assay, enzyme activity Enzymes mediate a wide range of cellular processes, and dynamic control

Heller, Eric

232

Development and validation of an in vitro micronucleus assay platform in TK6 cells.  

PubMed

The Organization for Economic Co-operation and Development (OECD) has recently adopted Test Guideline 487 (TG487) for conducting the in vitro micronucleus (MNvit) assay. The purpose of this study is to evaluate and validate treatment conditions for the use of p53 competent TK6 human lymphoblastoid cells in a TG487 compliant MNvit assay. The ten reference compounds suggested in TG487 (mitomycin C, cytosine arabinoside, cyclophosphamide, benzo-a-pyrene, vinblastine sulphate, colchicine, sodium chloride, nalidixic acid and di(2-ethylhexyl)phthalate and pyrene) and noscapine hydrochloride were chosen for this study. In order to optimize the micronucleus response after treatment with some positive substances, we extended the recovery time after pulse treatment from 2 cell cycles recommended in TG487 to 3 cell cycles for untreated cells (40h). Each compound was tested in at least one of four exposure conditions: a 4h exposure followed by a 40h recovery, a 4h exposure followed by a 24h recovery, a 4h exposure in the presence of an exogenous metabolic activation system followed by a 40h recovery period, and a 27h continuous direct treatment. Results show that the direct acting clastogens, clastogens requiring metabolic activation and aneugens caused a robust increase in micronuclei in at least one test condition whereas the negative compounds did not induce micronuclei. The negative control cultures exhibited reproducibly low and consistent micronucleus frequencies ranging from 0.4 to 1.8% (0.8±0.3% average and standard deviation). Furthermore, extending the recovery period from 24h to 40h produced a 2-fold higher micronucleus frequency after a 4h pulse treatment with mitomycin C. In summary, the protocol described in this study in TK6 cells produced the expected result with model compounds and should be suitable for performing the MNvit assay in accordance with guideline TG487. PMID:22445949

Sobol, Zhanna; Homiski, Michael L; Dickinson, Donna A; Spellman, Richard A; Li, Dingzhou; Scott, Andrew; Cheung, Jennifer R; Coffing, Stephanie L; Munzner, Jennifer B; Sanok, Kelley E; Gunther, William C; Dobo, Krista L; Schuler, Maik

2012-07-01

233

Microfluidic cell culture systems with integrated sensors for drug screening  

NASA Astrophysics Data System (ADS)

Cell-based testing is a key step in drug screening for cancer treatments. A microfluidic platform can permit more precise control of the cell culture microenvironment, such as gradients in soluble factors. These small-scale devices also permit tracking of low cell numbers. As a new screening paradigm, a microscale system for integrated cell culture and drug screening promises to provide a simple, scalable tool to apply standardized protocols used in cellular response assays. With the ability to dynamically control the microenvironment, we can create temporally varying drug profiles to mimic physiologically measured profiles. In addition, low levels of oxygen in cancerous tumors have been linked with drug resistance and decreased likelihood of successful treatment and patient survival. Our work also integrates a thin-film oxygen sensor with a microfluidic oxygen gradient generator which will in future allow us to create spatial oxygen gradients and study effects of hypoxia on cell response to drug treatment. In future, this technology promises to improve cell-based validation in the drug discovery process, decreasing the cost and increasing the speed in screening large numbers of compounds.

Grist, Samantha; Yu, Linfen; Chrostowski, Lukas; Cheung, Karen C.

2012-03-01

234

Detection of Mycoplasma contamination in cell cultures.  

PubMed

Mycoplasma contamination of cell lines is a major problem in cell culture technology. This unit presents protocols involving either the polymerase chain reaction (PCR) or fluorescent in situ hybridization (FISH) to provide independent, fast, and sensitive techniques to monitor mycoplasma contamination in laboratory cultures. Special emphasis is placed on the integration of control reactions to prevent false-negative as well as false-positive results due to reaction inhibition or contamination and background staining, respectively. Curr. Protoc. Mol. Biol. 106:28.4.1-28.4.14. © 2014 by John Wiley & Sons, Inc. PMID:24733240

Uphoff, Cord C; Drexler, Hans G

2014-01-01

235

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

236

Patterned cell culture substrates created by hot embossing of tissue culture treated polystyrene.  

PubMed

Patterning materials such that they elicit a different cell response in different regions would have significant implications in fields such as implantable biomaterials, in vitro cell culture and tissue engineering and regenerative medicine. Moreover, the ability to pattern polymers using inexpensive, currently available processes, without the need for adding proteins or other biochemical agents could lead to new opportunities in biomaterials research. The research reported here demonstrates that by combining the plasma surface treatments used to create commercial grade tissue culture treated polystyrene, with controlled hot embossing processes, that distinct regions can be created on a substrate that result in spatial control of endothelial cell adhesion and proliferation. As well as the topographical changes that result from hot embossing, significant changes in surface chemistry and wettability have been observed and characterised and the resultant effects on endothelial cell responses evaluated. By spatially controlling endothelial cell adhesion, proliferation and subsequent angiogenesis, the processes outlined here have the potential to be used to create a range of different substrates, with applications in the development of assays for high throughput screening, the patterning of implantable biomaterials or the development of smart scaffolds for tissue engineering. PMID:23900705

Brown, Alan; Burke, George A; Meenan, Brian J

2013-12-01

237

Establishment and assessment of a new human embryonic stem cell-based biomarker assay for developmental toxicity screening.  

PubMed

A metabolic biomarker-based in vitro assay utilizing human embryonic stem (hES) cells was developed to identify the concentration of test compounds that perturbs cellular metabolism in a manner indicative of teratogenicity. This assay is designed to aid the early discovery-phase detection of potential human developmental toxicants. In this study, metabolomic data from hES cell culture media were used to assess potential biomarkers for development of a rapid in vitro teratogenicity assay. hES cells were treated with pharmaceuticals of known human teratogenicity at a concentration equivalent to their published human peak therapeutic plasma concentration. Two metabolite biomarkers (ornithine and cystine) were identified as indicators of developmental toxicity. A targeted exposure-based biomarker assay using these metabolites, along with a cytotoxicity endpoint, was then developed using a 9-point dose-response curve. The predictivity of the new assay was evaluated using a separate set of test compounds. To illustrate how the assay could be applied to compounds of unknown potential for developmental toxicity, an additional 10 compounds were evaluated that do not have data on human exposure during pregnancy, but have shown positive results in animal developmental toxicity studies. The new assay identified the potential developmental toxicants in the test set with 77% accuracy (57% sensitivity, 100% specificity). The assay had a high concordance (?75%) with existing in vivo models, demonstrating that the new assay can predict the developmental toxicity potential of new compounds as part of discovery phase testing and provide a signal as to the likely outcome of required in vivo tests. PMID:24123775

Palmer, Jessica A; Smith, Alan M; Egnash, Laura A; Conard, Kevin R; West, Paul R; Burrier, Robert E; Donley, Elizabeth L R; Kirchner, Fred R

2013-08-01

238

Functional assay of the mutant tissue-nonspecific alkaline phosphatase gene using U2OS osteoblast-like cells.  

PubMed

Tissue-nonspecific alkaline phosphatase (TNAP) plays a key role in mineralization. A defect in the TNAP gene causes hypophosphatasia, which is characteristic of systemic skeletal hypomineralization. To determine the mineralizing ability of the mutant proteins, we developed a functional assay that uses U2OS osteoblast-like cells. Expression plasmids containing TNAP mutant cDNAs were constructed and introduced into U2OS cells, which are derived from a human osteosarcoma and exhibit very low alkaline phosphatase (ALP) activity and disabled mineralization. U2OS cells, in which active TNAP cDNAs were introduced, expressed high ALP activity and mineralized their circumstance when they were cultured with beta-glycerophosphate. The ALP activity in these U2OS cells corresponded to the activity reported for COS cells in which active TNAP cDNA was introduced. An in vitro mineralization assay of U2OS cells transfected with moderate allele cDNAs showed that approximately 35% of TNAP enzymatic activity may be the threshold value for mineralization. In addition, U2OS cells transfected with wild-type TNAP and polymorphism TNAP cDNA showed PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) induction as in SaOS-2 cells. In summary, the introduction of active TNAP cDNA into U2OS cells allowed these cells to mineralize, and this technique may be a useful functional assay of TNAP mutant proteins. PMID:18455459

Orimo, Hideo; Goseki-Sone, Masae; Hosoi, Takayuki; Shimada, Takashi

2008-07-01

239

Cell culture conditions affect RPE phagocytic function  

Microsoft Academic Search

Background  Changes in the phenotype of retinal pigment epithelium (RPE) cells in vitro are associated with medium conditions and changes\\u000a in function. Main goals in RPE tissue engineering are cell propagation in serum-free defined culture conditions, resulting\\u000a in cells exhibiting differentiated morphology and functioning in vitro.\\u000a \\u000a \\u000a \\u000a Methods  To compare the effects of various media and supplements on cell function, an optimized high-throughput

Mike O. Karl; Monika Valtink; Jürgen Bednarz; Katrin Engelmann

2006-01-01

240

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

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

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 ...

243

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

244

Cell growth assay using the ultrasonic flexural plate-wave device  

NASA Astrophysics Data System (ADS)

The ability to easily monitor the rate of cell growth has many useful applications such as characterizing cell lines to optimize growth conditions and compound screening to test for cytotoxicity. As a cell growth sensor, the ultrasonic flexural plate-wave device offers a convenient assay that is non-invasive, continuous, and capable of full automation for high throughput screening. The flexural plate-wave (FPW) device is a microfabricated sensor consisting of a thin silicon nitride membrane with aluminum and zinc oxide transducers at both ends. These interdigitated transducers send and receive acoustic plate waves that propagate along the membrane. Applications of the device include mass sensing, achieved by measuring the frequency shift caused by mass loading of the membrane, and pumping of liquids and gases. We have conducted studies that established the biocompatibility of the FPW device materials with cell cultures. In addition, biological samples were exposed to the ultrasonic agitation of the device and no damaging effects were found. Experiments were performed using the FPW device to sense cell growth for mammalian suspension (non- adherent) cells. Results show a good correlation between change of cell number and frequency.

Meng, Audra H.; White, Richard M.

1997-03-01

245

Hormonal growth control of cells in culture  

Microsoft Academic Search

Summary  Serum is the last undefined component in cell culture media. Our results indicate that the primary role of serum is to provide\\u000a hormones and that serum can be replaced by a group of hormones. A rat pituitary cell line, GH3, can grow in serum-free medium if the medium is supplemented with 3,3?,5-triiodothyronine, TSH-releasing hormone, transferrin,\\u000a parathyroid hormone, insulin and three

I. Hayashi; J. Larner; G. Sato

1978-01-01

246

Human T cell priming assay: depletion of peripheral blood lymphocytes in CD25(+) cells improves the in vitro detection of weak allergen-specific T cells.  

PubMed

To develop an in vitro assay that recapitulates the key event of allergic contact dermatitis (ACD), that is the priming of effector T cells by hapten-presenting dendritic cells, and then allows for the sensitive detection of chemical allergens represents a major challenge. Classical human T cell priming assays (hTCPA) that have been developed in the past, using hapten-loaded monocyte-derived dendritic cells (MDDCs) as antigen-presenting cells and peripheral blood lymphocytes (PBLs) as responding cells, were not efficient to prime T cells to common allergens with moderate/weak sensitizing properties. Recent progress in the understanding of the effector and regulatory mechanisms of ACD have shown that T cell priming requires efficient uptake of allergens by immunogenic DCs and that it is controlled by several subsets of regulatory cells including CD25(+) Tregs. We therefore analyzed various parameters involved in allergen-specific T cell activation in vitro and showed that priming of allergen-specific T cells is hampered by several subsets of immune cells comprising CD1a(neg) DCs, CD25(+) T cells, and CD56(+) regulatory cells.CD4(+)CD25(+)FoxP3(+) Tregs prevented the in vitro T cell priming to moderate/weak allergens, and depletion of human PBLs in CD25(+) cells significantly increased specific T cell proliferation and IFN-? secretion. CD56(+) cells exerted an additional control of T cell priming since co-depletion of both CD56(+) and CD25(+) cells improved the magnitude of chemical-specific T cell activation. Finally, CD1a(low) MDDCs were able to inhibit T cell activation obtained by allergen-pulsed CD1a(high) MDDC. Moreover, we showed that uptake by DC of allergen-encapsulated nanoparticles significantly increased their activation status and their ability to prompt specific T cell activation. Hence, by combining the different strategies, i.e., depletion of CD25(+) and CD56(+) cells, use of CD1a(high) MDDC, and nanoparticle encapsulation of allergens, it was possible to induce T cell priming to most of the moderate/weak allergens, including lipophilic molecules highly insoluble in culture media. Therefore, the present optimized in vitro human T cell priming assay is a valuable method to detect the sensitizing properties of chemical allergens. PMID:24214620

Vocanson, Marc; Achachi, Amine; Mutez, Virginie; Cluzel-Tailhardat, Magalie; Varlet, Béatrice Le; Rozières, Aurore; Fournier, Philippe; Nicolas, Jean-François

2014-01-01

247

How are pluripotent cells captured in culture?  

E-print Network

tetracyclin (Tet) inducible Oct3/4 transgene. Tet addition into the culture 1 media results in rapid loss of Oct3/4 protein and differentiation into 2 trophectoderm cells through de-repressing the Cdx2 and Eomesodermin 3 genes [78]. 4 5 5.2 Sox2 6 Sox2 is a...

Kinoshita, Masaki

2014-01-01

248

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

249

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

PubMed

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. PMID:24141454

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

2013-01-01

250

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

PubMed Central

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. PMID:24141454

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.

2013-01-01

251

Chemical Interrogation of the neuronal kinome using a primary cell-based screening assay  

PubMed Central

A fundamental impediment to functional recovery from spinal cord injury (SCI) and traumatic brain injury is the lack of sufficient axonal regeneration in the adult central nervous system. There is thus a need to develop agents that can stimulate axon growth to re-establish severed connections. Given the critical role played by protein kinases in regulating axon growth and the potential for pharmacological intervention, small molecule protein kinase inhibitors present a promising therapeutic strategy. Here, we report a robust cell-based phenotypic assay, utilizing primary rat hippocampal neurons, for identifying small molecule kinase inhibitors that promote neurite growth. The assay is highly reliable and suitable for medium throughput screening, as indicated by its Z?-factor of 0.73. A focused structurally diverse library of protein kinase inhibitors was screened, revealing several compound groups with the ability to strongly and consistently promote neurite growth. The best performing bioassay hit robustly and consistently promoted axon growth in a postnatal cortical slice culture assay. This study can serve as a jumping-off point for structure activity relationship (SAR) and other drug discovery approaches towards the development of drugs for treating SCI and related neurological pathologies. PMID:23480631

Al-Ali, Hassan; Schurer, Stephan C.; Lemmon, Vance P.; Bixby, John L.

2013-01-01

252

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

253

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.

Payne, Tiffany

2014-01-01

254

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

255

Methods to culture, differentiate, and characterize neural stem cells from the adult and embryonic mouse central nervous system.  

PubMed

Since the discovery of neural stem cells (NSC) in the embryonic and adult mammalian central nervous system (CNS), there have been a growing numbers of tissue culture media and protocols to study and functionally characterize NSCs and its progeny in vitro. One of these culture systems introduced in 1992 is referred to as the Neurosphere Assay, and it has been widely used to isolate, expand, differentiate and even quantify NSC populations. Several years later because its application as a quantitative in vitro assay for measuring NSC frequency was limited, a new single-step semisolid based assay, the Neural Colony Forming Cell (NCFC) assay was developed to accurately measure NSC numbers. The NCFC assay allows the discrimination between NSCs and progenitors by the size of colonies they produce (i.e., their proliferative potential). The evolution and continued improvements made to these tissue culture tools will facilitate further advances in the promising application of NSCs for therapeutic use. PMID:23179851

Louis, Sharon A; Mak, Carmen K H; Reynolds, Brent A

2013-01-01

256

Comparison of Loop-Mediated Isothermal Amplification Assay and Conventional Culture Methods for Detection of Campylobacter jejuni and Campylobacter coli in Naturally Contaminated Chicken Meat Samples  

Microsoft Academic Search

We investigated the efficacy of a loop-mediated isothermal amplification (LAMP) assay for detection of chicken meat samples naturally contaminated with Campylobacter jejuni and Campylobacter coli. A total of 144 Preston enrichment broth cultures from chicken meat samples were assessed by using the LAMP assay and conventional culture methods, which consist of a combination of Preston enrichment culturing and plating onto

Wataru Yamazaki; Masumi Taguchi; Takao Kawai; Kentaro Kawatsu; Junko Sakata; Kiyoshi Inoue; Naoaki Misawa

2009-01-01

257

A quick and low-cost PCR-based assay for Candida spp. identification in positive blood culture bottles  

PubMed Central

Background Differences in the susceptibility of Candida species to antifungal drugs make identification to the species level important for clinical management of candidemia. Molecular tests are not yet standardized or available in most clinical laboratories, although such tests can reduce the time required for species identification, as compared to the conventional culture-based methods. To decrease laboratory costs and improve diagnostic accuracy, different molecular methods have been proposed, including DNA extraction protocols to produce pure DNA free of PCR inhibitors. The objective of this study was to validate a new format of molecular method, based on the internal transcribed spacer (ITS) of the rDNA gene amplification followed by sequencing, to identify common and cryptic Candida species causing candidemia by analyzing DNA in blood culture bottles positive for yeasts. Methods For DNA extraction, an “in-house” protocol based on organic solvent extraction was tested. Additional steps of liquid nitrogen incubation followed by mechanical disruption ensured complete cell lysis, and highly pure DNA. One hundred sixty blood culture bottles positive for yeasts were processed. PCR assays amplified the ITS region. The DNA fragments of 152 samples were sequenced and these sequences were identified using the GenBank database (NCBI). Molecular yeast identification was compared to results attained by conventional method. Results The organic solvent extraction protocol showed high reproducibility in regards to DNA quantity, as well as high PCR sensitivity (10 pg of C. albicans DNA and 95% amplification on PCR). The identification of species at the molecular level showed 97% concordance with the conventional culturing method. The molecular method tested in the present study also allowed identification of species not commonly implicated in human infections. Conclusions This study demonstrated that our molecular method presents significant advantages over the conventional yeast culture identification method by providing accurate results within 24 hours, in contrast to at least 72 hours required by the automated conventional culture method. Additionally, our molecular method allowed the identification of mixed infections, as well as infections due to emergent fungal pathogens. This economical DNA extraction method developed in our laboratory provided high-quality DNA and 60% cost savings compared to commercial methods. PMID:24099320

2013-01-01

258

Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cultures in microgravity  

NASA Technical Reports Server (NTRS)

The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European 'Biorack' provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the 'Biorack' facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatant in-flight), injection port, and supernatant collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatant, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground-based investigations simulating the conditions expected in the flight experiment. Several parameters including cell concentration, time between cell loading and activation, and storage temperature on cell survival were examined to characterise cell response and optimise the experiments to be flown aboard the Space Shuttle. Results indicate that the objectives of the experiments could be met with delays up to 5 days between cell loading into the hardware and initial in flight experiment activation, without the need for medium exchange. Experiment hardware of this kind, which is adaptable to a wide range of cell types and can be easily interfaced to different spacecraft facilities, offers the possibility for a wide range of experimenters successfully and easily to utilise future flight opportunities.

Hatton, J. P.; Lewis, M. L.; Roquefeuil, S. B.; Chaput, D.; Cazenave, J. P.; Schmitt, D. A.

1998-01-01

259

Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cultures in microgravity.  

PubMed

The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European 'Biorack' provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the 'Biorack' facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatant in-flight), injection port, and supernatant collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatant, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground-based investigations simulating the conditions expected in the flight experiment. Several parameters including cell concentration, time between cell loading and activation, and storage temperature on cell survival were examined to characterise cell response and optimise the experiments to be flown aboard the Space Shuttle. Results indicate that the objectives of the experiments could be met with delays up to 5 days between cell loading into the hardware and initial in flight experiment activation, without the need for medium exchange. Experiment hardware of this kind, which is adaptable to a wide range of cell types and can be easily interfaced to different spacecraft facilities, offers the possibility for a wide range of experimenters successfully and easily to utilise future flight opportunities. PMID:9671231

Hatton, J P; Lewis, M L; Roquefeuil, S B; Chaput, D; Cazenave, J P; Schmitt, D A

1998-08-01

260

Effect of Dietary Inducer Dimethylfumarate on Glutathione in Cultured Human Retinal Pigment Epithelial Cells  

Microsoft Academic Search

PURPOSE. To determine the effect of dimethylfumarate (DMF), an inducer of glutathione (GSH)- dependent detoxification, on intracellular GSH levels in cultured human retinal pigment epithelium (hRPE) cells, its mechanism of action, and its effect on hRPE cells subjected to oxidative injury. METHODS. Established hRPE cell lines were treated with DMF and assayed by high-pressure liquid chromatography for intracellular and extracellular

Kasey C. Nelson; Joanne L. Carlson; Melanie L. Newman; Paul Sternberg; Dean P. Jones; Terrance J. Kavanagh; Dolores Diaz; Jiyang Cai; Mei Wu

261

Microinjection of Single Cells in Culture Louise Cramer December 1995  

E-print Network

, then 1x with culture media. 3. Plate cells. #12;Observation Media To observe cells on a microscope we use a media optimal for preserving cell health. Maintaining pH outside of a tissue culture incubator is mostMicroinjection of Single Cells in Culture Louise Cramer December 1995 Introduction We typically

Mitchison, Tim

262

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

263

Plant cell cultures: bioreactors for industrial production.  

PubMed

The recent biotechnology boom has triggered increased interest in plant cell cultures, since a number of firms and academic institutions investigated intensively to rise the production of very promising bioactive compounds. In alternative to wild collection or plant cultivation, the production of useful and valuable secondary metabolites in large bioreactors is an attractive proposal; it should contribute significantly to future attempts to preserve global biodiversity and alleviate associated ecological problems. The advantages of such processes include the controlled production according to demand and a reduced man work requirement. Plant cells have been grown in different shape bioreactors, however, there are a variety of problems to be solved before this technology can be adopted on a wide scale for the production of useful plant secondary metabolites. There are different factors affecting the culture growth and secondary metabolite production in bioreactors: the gaseous atmosphere, oxygen supply and CO2 exchange, pH, minerals, carbohydrates, growth regulators, the liquid medium rheology and cell density. Moreover agitation systems and sterilization conditions may negatively influence the whole process. Many types ofbioreactors have been successfully used for cultivating transformed root cultures, depending on both different aeration system and nutrient supply. Several examples of medicinal and aromatic plant cultures were here summarized for the scale up cultivation in bioreactors. PMID:21520713

Ruffoni, Barbara; Pistelli, Laura; Bertoli, Alessandra; Pistelli, Luisa

2010-01-01

264

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

265

Hepatotoxicity of salicylates in monolayer cell cultures.  

PubMed

The influence of graded doses of sodium salicylate on rat liver cells cultured in monolayers was assessed by measuring lactic dehydrogenase activity in a culture media after incubation. Morphological alterations were studied by electron microscopy. The influence of different albumin concentrations in the media on toxicity was also evaluated. Lactic dehydrogenase concentrations rose with increasing doses of salicylate up to 40 mg per dl. High concentrations of albumin were associated with reduced salicylate toxicity. These findings suggest that salicylate-induced hepatic injury is dose related and my be influenced by serum albumin levels PMID:620893

Tolman, K G; Peterson, P; Gray, P; Hammar, S P

1978-02-01

266

Development of an intestinal cell culture model to obtain smooth muscle cells and myenteric neurones  

PubMed Central

This paper reports on the development of an entirely new intestinal smooth muscle cell (ISMC) culture model using rat neonates for use in pharmacological research applications. Segments of the duodenum, jejunum and ileum were obtained from Sprague-Dawley rat neonates. The cell extraction technique consisted of ligating both ends of the intestine and incubating (37 ºC) in 0.25% trypsin for periods of 30–90 min. Isolated cells were suspended in DMEM-HEPES, plated and allowed to proliferate for 7 days. Cell culture quality was assessed via a series of viability tests using the dye exclusion assay. In separate experiments, tissues were exposed to trypsin for varying durations and subsequently histological procedures were applied. Cell purification techniques included differential adhesion technique for minimizing fibroblasts. Selective treatments with neurotoxin scorpion venom (30 µg mL?1) and anti-mitotic cytosine arabinoside (6 µm) were also applied to purify respectively ISMC and myenteric neurones selectively. The different cell populations were identified in regard to morphology and growth characteristics via immunocytochemistry using antibodies to smooth muscle ?-actin, ?-actinin and serotonin-5HT3 receptors. Based on both viability and cell confluence experiments, results demonstrated that intestinal cells were best obtained from segments of the ileum dissociated in trypsin for 30 min. This provided the optimum parameters to yield highly viable cells and confluent cultures. The finding was further supported by histological studies demonstrating that an optimum incubation time of 30 min is required to isolate viable cells from the muscularis externae layer. When cell cultures were treated with cytosine arabinoside, the non-neuronal cells were abolished, resulting in the proliferation of cell bodies and extended neurites. Conversely, cultures treated with scorpion venom resulted in complete abolition of neurones and proliferation of increasing numbers of ISMC, which were spindle-shaped and uniform throughout the culture. When characterized by immunocytochemistry, neurones were stained with antibody to 5HT3 receptors but not with antibodies to ?-smooth muscle actin and ?-actinin. Conversely, ISMC were stained with antibodies to ?-smooth muscle actin and ?-actinin but not with antibody to 5HT3 receptors. The present study provides evidence that our method of dissociation and selectively purifying different cell populations will allow for pharmacological investigation of each cell type on different or defined mixtures of different cell types. PMID:17979953

Batista Lobo, S; Denyer, M; Britland, S; Javid, F A

2007-01-01

267

Effect of methisoprinol on virus replication in cell cultures.  

PubMed

The effect of Methisoprinol (active substance: isoprinozine) on the replication of two animal viruses, the TK900 strain of Aujeszky's disease virus and the Roakin strain of the Newcastle disease virus was investigated. When the maximal tolerable doses of the drug were added to two cell cultures (CECC and GMK), its effect on the level of infectious titres of theviruses and their adsorption were assayed. Investigations were also performed to assess the direct effect of Methisoprinol on the viral strains used. The final stage of the experiment aimed at analysing of the replication dynamics of the viruses in the presence of Methisoprinol. Methisoprinol showed no direct effect on the viruses used in the study. Nor did it affect their adsorption. The preparation applied to the culture 24 hours before infection did not influence the replication of viruses, but administered simultaneously with the infection significantly lowered the final titres of viruses. The highest inhibitory effect of the drug was observed during the analysis of the replication dynamics of both viruses in CECC and of pseudorabies virus in GMK cell culture upon the application of the maximal tolerable doses of Methisoprinol and low infectious doses of the viruses. PMID:15230539

Ma?aczewska, J; Rotkiewicz, Z

2004-01-01

268

Subacute cytotoxicity testing with cultured human lung cells.  

PubMed

This study was designed to evaluate the potential of an in vitro cell culture method for its ability to determine subacute cytotoxicity and to compare the cytotoxic concentrations with rodent LD(50)s and clinical human toxicity data. Human fetal lung fibroblasts (HFL1) were incubated in the absence or presence of increasing concentrations of test chemicals for 72 h, and cell proliferation was used as a marker for toxicity. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis. Comparison of the cytotoxicity data from testing 50 chemicals, with available human lethal concentrations for the same chemicals, revealed that the 72-h experimental IC(50)s are as accurate predictors of human toxicity as equivalent toxic blood concentrations derived from rodent LD(50)s. In addition, our results demonstrate that subacute 72-h exposure of HFL1 cells more accurately predicts cytotoxicity than a 24-h mitochondrial assay previously conducted in our laboratory, although the experimental IC(50) values were not statistically different in the two assays. It is anticipated that this procedure, together with a related battery of tests, may supplement or replace currently used animal protocols to screen chemicals for human risk assessment. PMID:11812637

Yang, A; Cardona, D L; Barile, F A

2002-02-01

269

Metabolic stability of experimental chemotherapeutic agents in hepatocyte:tumor cell co-cultures.  

PubMed

A U.S. National Cancer Institute screening program for new anticancer drugs, based on the growth of primary human tumor cells in an in vitro soft agar colony formation assay, has resulted in the identification of a number of compounds that have cytotoxic activity against primary human tumor cells in vitro but are inactive in the conventional in vivo murine P388 leukemia animal model pre-screen. To investigate whether metabolic inactivation ov the compounds might be a factor in the lack of in vivo cytotoxicity we have co-cultured rat hepatocytes with A204 rhabdomyosarcoma and murine P388 leukemia cell lines in the soft agarose colony formation assay for 24 h during exposure to the compounds. Twenty compounds with a range of in vitro activities were studied. Thirteen compounds exhibited cytotoxicity against A204 cells in culture; nine of them were less active when co-cultured with hepatocytes, two were activated by hepatocyte co-culture, and two showed no effect of hepatocyte co-culture. P388 cells were more sensitive to the antiproliferative effects of the compounds than A204 cells. Two compounds that were not active against A204 cells exhibited cytotoxicity against P388 cells. One compound was inactivated by hepatocyte co-culture and one showed no effect. Five compounds showed no cytotoxicity toward either A204 cells or P388 cells. Two of the compounds showing hepatocyte inactivation in vitro possess activity in one or more in vivo tumor models. Thus, evidence for metabolic inactivation in hepatocyte co-culture is not always an indication for lack of in vivo antitumor activity. Hepatocyte co-culture methodology provides a simple and objective means, amenable to large-scale screening, of distinguishing metabolic activation or inactivation of a given compound from other pharmacokinetic and pharmacodynamic factors with a minimum of material. PMID:3698177

Appel, P L; Alley, M C; Lieber, M M; Shoemaker, R; Powis, G

1986-01-01

270

The birth of therapy with cultured cells.  

PubMed

Long ago, I set out to solve a problem, but something happened along the way: I was diverted by an unexpected observation. Thereafter, the direction of my research was guided at each stage by increasing familiarity with the experimental material and what could be done with it. The result was the birth of therapy with cultured keratinocytes. Subsequent developments soon led to the formation of the company Biosurface Technology (later taken over by the Genzyme Corporation), which provided autologous cultures for burn victims in many parts of the world. Further progress by others led to new therapeutic applications of cultured keratinocytes, such as treatment of an ocular disease and gene therapy. Unfortunately, there have developed serious regulatory problems that are a danger to future progress. As described in this brief history, the initial stages of development of cell therapy for the treatment of human disease were possible only because there was no restraint by committees or governmental regulations. PMID:18693268

Green, Howard

2008-09-01

271

Tubulin dynamics in cultured mammalian cells  

PubMed Central

Bovine neurotubulin has been labeled with dichlorotriazinyl- aminofluorescein (DTAF-tubulin) and microinjected into cultured mammalian cells strains PTK1 and BSC. The fibrous, fluorescence patterns that developed in the microinjected cells were almost indistinguishable from the pattern of microtubules seen in the same cells by indirect immunofluorescence. DTAF-tubulin participated in the formation of all visible, microtubule-related structures at all cell cycle stages for at least 48 h after injection. Treatments of injected cells with Nocodazole or Taxol showed that DTAF-tubulin closely mimicked the behavior of endogenous tubulin. The rate at which microtubules incorporated DTAF-tubulin depended on the cell-cycle stage of the injected cell. Mitotic microtubules became fluorescent within seconds while interphase microtubules required minutes. Studies using fluorescence redistribution after photobleaching confirmed this apparent difference in tubulin dynamics between mitotic and interphase cells. The temporal patterns of redistribution included a rapid phase (approximately 3 s) that we attribute to diffusion of free DTAF-tubulin and a second, slower phase that seems to represent the exchange of bleached DTAF-tubulin in microtubules with free, unbleached DTAF- tubulin. Mean half times of redistribution were 18-fold shorter in mitotic cells than they were in interphase cells. PMID:6501419

1984-01-01

272

Eradication of Mycoplasma contaminations from cell cultures.  

PubMed

Mycoplasma contaminations have a multitude of effects on cultured cell lines that may influence the results of experiments or pollute bioactive substances isolated from the eukaryotic cells. The elimination of mycoplasma contaminations from cell cultures with antibiotics has been proven to be a practical alternative to discarding and re-establishing important or irreplaceable cell lines. Different fluoroquinolones, tetracyclins, pleuromutilins, and macrolides shown to have strong anti-mycoplasma properties are employed for the decontamination. These antibiotics are applied as single treatments, as combination treatment of two antibiotics in parallel or successively, or in combination with a surface-active peptide to enhance the action of the antibiotic. The protocols in this unit allow eradication of mycoplasmas, prevention of the development of resistant mycoplasma strains, and potential cure of heavily contaminated and damaged cells. Consistent and permanent alterations to eukaryotic cells attributable to the treatment have not been demonstrated. Curr. Protoc. Mol. Biol. 106:28.5.1-28.5.12. © 2014 by John Wiley & Sons, Inc. PMID:24733241

Uphoff, Cord C; Drexler, Hans G

2014-01-01

273

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

274

Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices.  

PubMed

Culture of cells using various microfluidic devices is becoming more common within experimental cell biology. At the same time, a technological radiation of microfluidic cell culture device designs is currently in progress. Ultimately, the utility of microfluidic cell culture will be determined by its capacity to permit new insights into cellular function. Especially insights that would otherwise be difficult or impossible to obtain with macroscopic cell culture in traditional polystyrene dishes, flasks or well-plates. Many decades of heuristic optimization have gone into perfecting conventional cell culture devices and protocols. In comparison, even for the most commonly used microfluidic cell culture devices, such as those fabricated from polydimethylsiloxane (PDMS), collective understanding of the differences in cellular behavior between microfluidic and macroscopic culture is still developing. Moving in vitro culture from macroscopic culture to PDMS based devices can come with unforeseen challenges. Changes in device material, surface coating, cell number per unit surface area or per unit media volume may all affect the outcome of otherwise standard protocols. In this review, we outline some of the advantages and challenges that may accompany a transition from macroscopic to microfluidic cell culture. We focus on decisive factors that distinguish macroscopic from microfluidic cell culture to encourage a reconsideration of how macroscopic cell culture principles might apply to microfluidic cell culture. PMID:25105943

Halldorsson, Skarphedinn; Lucumi, Edinson; Gómez-Sjöberg, Rafael; Fleming, Ronan M T

2015-01-15

275

Rapid Detection of Methicillin-Resistant Staphylococci from Blood Culture Bottles by Using a Multiplex PCR Assay  

PubMed Central

Rapid detection and accurate identification of methicillin-resistant staphylococci are critical for the effective management of infections caused by these organisms. We describe a multiplex PCR-based assay for the direct detection of methicillin-resistant staphylococci from blood culture bottles (BacT/Alert; Organon-Teknika, Durham, N.C.). A simple lysis method followed by a multiplex PCR assay designed to detect the nuc, mecA, and bacterial 16S rRNA genes was performed. A total of 306 blood culture specimens were collected over a period of 10 months from June 1998 to April 1999, consisting of 236 blood cultures growing staphylococci (including 124 methicillin-resistant Staphylococcus spp.), 50 positive blood cultures which grew organisms other than staphylococci, and 20 blood cultures that were negative for bacterial and fungal pathogens after 5 days of incubation and terminal subculture. DNA extraction, PCR, and detection could be completed in 2.5 h. Of the positive blood cultures with staphylococci, the multiplex PCR assay had a sensitivity and specificity of 99.2% and 100%, respectively. Our results show that rapid, direct detection of methicillin-resistant staphylococci is possible, allowing clinicians to make prompt and effective decisions for the management of patients with staphylococcal bacteremia. PMID:12149330

Louie, L.; Goodfellow, J.; Mathieu, P.; Glatt, A.; Louie, M.; Simor, A. E.

2002-01-01

276

Behaviour of cell cultures from human amniotic fluid  

Microsoft Academic Search

The growth pattern of cell cultures originating from 11 amniotic fluid specimens have been observed. From each specimen 2 to 12 primary cultures were set up. In most cases growth started simultaneously in the primary cultures originating from one sample. The primary cultures lasted from 7 to 30 days. A variation was found both between cultures from different pregnancies as

L Hasholt

1976-01-01

277

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

278

21 CFR 864.2280 - Cultured animal and human cells.  

Code of Federal Regulations, 2012 CFR

... FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a)...

2012-04-01

279

21 CFR 864.2280 - Cultured animal and human cells.  

... FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a)...

2014-04-01

280

21 CFR 864.2280 - Cultured animal and human cells.  

Code of Federal Regulations, 2013 CFR

... FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a)...

2013-04-01

281

Eyes Wide Open: A Critical Review of Sphere-Formation as an Assay For Stem Cells  

PubMed Central

Sphere-forming assays have been widely used to retrospectively identify stem cells based on their reported capacity to evaluate self-renewal and differentiation at the single cell level in vitro. The discovery of markers that allow the prospective isolation of stem cells and their progeny from their in vivo niche allows the functional properties of purified populations to be defined. We provide an historical perspective of the evolution of the neurosphere assay, and highlight limitations in the use of sphere-forming assays, in the context of neurospheres. We discuss theoretical and technical considerations of experimental design and interpretation that surround the use of this assay with any tissue. PMID:21549325

Pastrana, Erika; Silva-Vargas, Violeta; Doetsch, Fiona

2012-01-01

282

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

283

Comparative study with two different enrichments in the culture media used in the disinfectant efficacy assay.  

PubMed

Recent changes in Brazilian legislation for commercial disinfectants have been published due to the recent epidemic of nosocomial infections caused by rapidly growing mycobacteria (RGM) in many states of Brazil over the last 8years. One of these documents requires that all the manufacturers provide evidence of efficacy of sterilizing and disinfectant products, used for semi critical medical devices, against the Mycobacterium bovis BCG Moreau and Mycobacterium abscessus subsp. bolletii INCQS 00594 strains by using the Confirmative in vitro Test for Determining Tuberculocidal Activity of Disinfectants recommended by the Association of Official Analytical Chemists. These changes have caused additional costs and increased problems for importation of enrichment products at national laboratories where disinfectant efficacy assay service is performed. Middlebrook ADC Enrichment (ADC) is provided by a unique manufacturer and used in the official protocol. The aim of the present study was to evaluate an alternative in house low-cost enrichment composed of fetal bovine serum and glucose (FBSG) with ADC for performance of disinfectant efficacy assay against mycobacteria. After obtaining the growth curves for M. abscessus ATCC 19977, M. abscessus subsp. bolletii INCQS 00594, Mycobacterium chelonae ATCC 35752, and Mycobacterium fortuitum ATCC 6841 by using ADC enrichment and FBSG in Kirchners and 7H9 culture media. Through statistical analysis via the Kruskal-Wallis test on the evaluation of microorganism growth rate, it was observed that there was no inhibition of RGM growth by any of the enrichments used. These results suggest that low-cost enrichment FBSG may be used as a potential substitute of ADC for composition of media for mycobacterial growth, including in disinfectant tests. PMID:22197720

Sabagh, Bruna Peres; Souto, Aline da Silva Soares; Reis, Louise Moreira; Silva, Sérgio Alves da; Pereira, Daniella Cristina Rodrigues; Neves, Marta de Campos; Pinheiro, Rodrigo Rollin; Duarte, Rafael Silva; Miyazaki, Neide Hiromi Tokumaru; Bôas, Maria Helena Simões Villas

2012-02-01

284

Production of avian influenza virus vaccine using primary cell cultures generated from host organs.  

PubMed

The global availability of a therapeutically effective influenza virus vaccine during a pandemic remains a major challenge for the biopharmaceutical industry. Long production time, coupled with decreased supply of embryonated chicken eggs (ECE), significantly affects the conventional vaccine production. Transformed cell lines have attained regulatory approvals for vaccine production. Based on the fact that the avian influenza virus would infect the cells derived from its natural host, the viral growth characteristics were studied on chicken embryo-derived primary cell cultures. The viral propagation was determined on avian origin primary cell cultures, transformed mammalian cell lines, and in ECE. A comparison was made between these systems by utilizing various cell culture-based assays. In-vitro substrate susceptibility and viral infection characteristics were evaluated by performing hemagglutination assay (HA), 50 % tissue culture infectious dose (TCID??) and monitoring of cytopathic effects (CPE) caused by the virus. The primary cell culture developed from chicken embryos showed stable growth characteristics with no contamination. HA, TCID??, and CPE exhibited that these cell systems were permissive to viral infection, yielding 2-10 times higher viral titer as compared to mammalian cell lines. Though the viral output from the ECE was equivalent to the chicken cell culture, the time period for achieving it was decreased to half. Some of the prerequisites of inactivated influenza virus vaccine production include generation of higher vial titer, independence from exogenous sources, and decrease in the production time lines. Based on the tests, it can be concluded that chicken embryo primary cell culture addresses these issues and can serve as a potential alternative for influenza virus vaccine production. PMID:23515853

Babar, Mustafeez Mujtaba; Riaz, Muhammad Suleman; Zaidi, Najam-us-Sahar Sadaf; Afzal, Farhan; Farooq, Muhammad Sabir

2013-06-01

285

Natural products from plant cell cultures  

Microsoft Academic Search

Plants produce complex small molecules — natural products — that exhibit anticancer, antimalarial and antimicrobial activity.\\u000a These molecules play a key role in human medicine. However, plants typically produce these compounds in low quantities, and\\u000a harvesting plant natural products is frequently expensive, time-consuming and environmentally damaging. Plant cell culture\\u000a provides a renewable, easily scalable source of plant material. In this

Elizabeth McCoy; Sarah E. O’Connor

286

Striatal interneurons in dissociated cell culture  

Microsoft Academic Search

In addition to the well-characterized direct and indirect projection neurons there are four major interneuron types in the\\u000a striatum. Three contain GABA and either parvalbumin, calretinin or NOS\\/NPY\\/somatostatin. The fourth is cholinergic. It might\\u000a be assumed that dissociated cell cultures of striatum (typically from embryonic day E18.5 in rat and E14.5 for mouse) contain\\u000a each of these neuronal types. However,

S. C. Schock; K. S. Jolin-Dahel; P. C. Schock; W. A. Staines; M. Garcia-Munoz; Gordon W. Arbuthnott

2010-01-01

287

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

288

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

289

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

290

Ketamine is toxic to chondrocyte cell cultures.  

PubMed

Ketamine has been used in combination with a variety of other agents for intra-articular analgesia, with promising results. However, although it has been shown to be toxic to various types of cell, there is no available information on the effects of ketamine on chondrocytes. We conducted a prospective randomised controlled study to evaluate the effects of ketamine on cultured chondrocytes isolated from rat articular cartilage. The cultured cells were treated with 0.125 mM, 0.250 mM, 0.5 mM, 1 mM and 2 mM of ketamine respectively for 6 h, 24 hours and 48 hours, and compared with controls. Changes of apoptosis were evaluated using fluorescence microscopy with a 490 nm excitation wavelength. Apoptosis and eventual necrosis were seen at each concentration. The percentage viability of the cells was inversely proportional to both the duration and dose of treatment (p = 0.002 and p = 0.009). Doses of 0.5 mM, 1 mM and 2mM were absolutely toxic. We concluded that in the absence of solid data to support the efficacy of intra-articular ketamine for the control of pain, and the toxic effects of ketamine on cultured chondrocytes shown by this study, intra-articular ketamine, either alone or in combination with other agents, should not be used to control pain. Cite this article: Bone Joint J 2014; 96-B:989-94. PMID:24986956

Ozturk, A M; Ergun, M A; Demir, T; Gungor, I; Yilmaz, A; Kaya, K

2014-07-01

291

Use of Frozen Chick and Duck Embryo Cells for Plaque Assays of Arboviruses and Rickettsiae.  

National Technical Information Service (NTIS)

Conditions for preparation, freezing, storing and plating of chick and duck embryo cell slurries are described. Comparisons of plaque assay sensitivity for fresh and previously frozen cells with Eastern equine encephalitis, Western equine encephalitis, Ve...

A. T. McManus, D. R. Parker, R. H. Kenyon, J. P. Kondig, G. A. Eddy

1974-01-01

292

Triethyllead treatment of cultured brain cells. Effect on accumulation of radioactive precursors in galactolipids  

SciTech Connect

Cultured cells from chick embryo brains were studied for their sensitivity to triethyllead. Triethyllead chloride (3.16 microM) was added to the nutrient medium and incubated for 48 hr with the cells. Morphological changes in light microscope and radioactive labeling of galactolipids were assayed. Triethyllead treatment reduced the number of neuronal cells with processes. Morphological changes were not observed in glial cells. The (/sup 35/S)sulfate labeling of sulfatides was reduced to 50%. The (/sup 3/H)serine labeling of cerebrosides with alpha-hydroxy fatty acids was not influenced, while the (/sup 3/H)serine labeling of cerebrosides with nonhydroxy fatty acids was inhibited 40% in one- and two- but not in three-week-old cultures. The results indicate that the nerve cell response to triethyllead in cultures is selective, since the neurons are more sensitive than the glia cells and the labeling of sulfatides is more sensitive than that of cerebrosides.

Grundt, I.K.; Ammitzboll, T.; Clausen, J.

1981-02-01

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

Induction of antibody to foot-and-mouth disease virus in presensitized mouse spleen cell cultures.  

PubMed Central

Cultures of spleen cells from immunized mice were stimulated in vitro by soluble preparations of purified foot-and-mouth disease virus. Virus-specific antibody, as detected by an enzyme-linked immunosorbent assay, was produced by immune spleen cells but not by normal, nonimmune cells. The optimal specific response was obtained with 1 microgram of virus per ml of culture; as the virus concentration was increased, the production of specific antibody was reduced. For very low concentrations of virus (less than 0.01 microgram per culture), there was tentative evidence of suppression of the specific antibody response. The levels of specific antibody induced were dependent on the source and number of plastic-adherent cells present in the cultures. We intend to use this model system to study further the basis of immunity to foot-and-mouth disease virus. PMID:6092687

Collen, T; McCullough, K C; Doel, T R

1984-01-01

295

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

296

An Introductory Undergraduate Course Covering Animal Cell Culture Techniques  

ERIC Educational Resources Information Center

Animal cell culture is a core laboratory technique in many molecular biology, developmental biology, and biotechnology laboratories. Cell culture is a relatively old technique that has been sparingly taught at the undergraduate level. The traditional methodology for acquiring cell culture training has been through trial and error, instruction when…

Mozdziak, Paul E.; Petitte, James N.; Carson, Susan D.

2004-01-01

297

Limiting Dilution Analysis of Murine Epidermal Stem Cells Using an In Vivo Regeneration Assay  

PubMed Central

Summary Epidermal stem cells are of major importance for tissue homeostasis, wound repair, tumor initiation, and gene therapy. Here we describe an in vivo regeneration assay to test for the ability of keratinocyte progenitors to maintain an epidermis over the long term in vivo. Limiting dilution analysis of epidermal repopulating units in this in vivo regeneration assay at sequential time points allows the frequency of short term (transit amplifying cell) and long term (stem cell) repopulating cells to be quantified. PMID:19908020

Strachan, Lauren R.; Ghadially, Ruby

2009-01-01

298

Ascorbic acid transport into cultured pituitary cells  

SciTech Connect

An amidating enzyme designated peptidyl-glycine ..cap alpha..-amidating monooxygenase (PAM) has been studied in a variety of tissues and is dependent on molecular oxygen and stimulated by copper and ascorbic acid. To continue investigating the relationship among cellular ascorbic acid concentrations, amidating ability, and PAM activity, the authors studied ascorbic acid transport in three cell preparations that contain PAM and produce amidated peptides: primary cultures of rat anterior and intermediate pituitary and mouse AtT-20 tumor cells. When incubated in 50 ..mu..M (/sup 14/C)ascorbic acid all three cell preparations concentrated ascorbic acid 20- to 40-fold, producing intracellular ascorbate concentrations of 1 to 2 mM, based on experimentally determined cell volumes. All three cell preparations displayed saturable ascorbic acid uptake with half-maximal initial rates occurring between 9 and 18 ..mu..M ascorbate. Replacing NaCl in the uptake buffer with choline chloride significantly diminished ascorbate uptake in all three preparations. Ascorbic acid efflux from these cells was slow, displaying half-lives of 7 hours. Unlike systems that transport dehydroascorbic acid, the transport system for ascorbic acid in these cells was not inhibited by glucose. Thus, ascorbate is transported into pituitary cells by a sodium-dependent, active transport system.

Cullen, E.I.; May, V.; Eipper, R.A.

1986-05-01

299

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

300

Copper Modulates the Differentiation of Mouse Hematopoietic Progenitor Cells in Culture  

PubMed Central

Copper chelation has been shown to favor the expansion of human hematopoietic stem/progenitor cells in vitro. To further understand the effects of copper modulation on defined subsets of stem cells versus progenitor cells, we extended the studies in a mouse system. We isolated mouse hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) and cultured them with or without the copper chelator tetraethylen-epentamine (TEPA) or CuCl2. Cytokine-stimulated HPC cultures treated with TEPA for 7 days generated about two to three times more total and erythroid colony-forming cells (CFCs) compared to control cultures. In contrast, CuCl2 treatment decreased the CFC numbers. Similar results were seen with HSC after 14, but not 7, days of culture. Transplant studies showed that HPCs cultured for 7 days in TEPA had about twofold higher short-term erythroid repopulation potential compared to control cultures, while CuCl2 decreased the erythroid potential of cultured HPCs compared to control cultures. HSCs cultured with TEPA for 7 days did not exhibit significantly higher repopulation potential in either leukocyte or erythrocyte lineages compared to control cultures in short-term or long-term assays. Based on JC-1 staining, the mitochondrial membrane potential of HPCs cultured with TEPA was lower relative to control cultures. Our data suggest that decreasing the cellular copper content with TEPA results in preferential expansion or maintenance of HPC that are biased for erythroid differentiation in vivo, but does not enhance the maintenance of HSC activity in culture. PMID:19520051

Huang, Xiaosong; Pierce, L. Jeanne; Cobine, Paul A.; Winge, Dennis R.; Spangrude, Gerald J.

2014-01-01

301

Survey of culture, goldengate assay, universal biosensor assay, and 16S rRNA Gene sequencing as alternative methods of bacterial pathogen detection.  

PubMed

Cultivation-based assays combined with PCR or enzyme-linked immunosorbent assay (ELISA)-based methods for finding virulence factors are standard methods for detecting bacterial pathogens in stools; however, with emerging molecular technologies, new methods have become available. The aim of this study was to compare four distinct detection technologies for the identification of pathogens in stools from children under 5 years of age in The Gambia, Mali, Kenya, and Bangladesh. The children were identified, using currently accepted clinical protocols, as either controls or cases with moderate to severe diarrhea. A total of 3,610 stool samples were tested by established clinical culture techniques: 3,179 DNA samples by the Universal Biosensor assay (Ibis Biosciences, Inc.), 1,466 DNA samples by the GoldenGate assay (Illumina), and 1,006 DNA samples by sequencing of 16S rRNA genes. Each method detected different proportions of samples testing positive for each of seven enteric pathogens, enteroaggregative Escherichia coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shigella spp., Campylobacter jejuni, Salmonella enterica, and Aeromonas spp. The comparisons among detection methods included the frequency of positive stool samples and kappa values for making pairwise comparisons. Overall, the standard culture methods detected Shigella spp., EPEC, ETEC, and EAEC in smaller proportions of the samples than either of the methods based on detection of the virulence genes from DNA in whole stools. The GoldenGate method revealed the greatest agreement with the other methods. The agreement among methods was higher in cases than in controls. The new molecular technologies have a high potential for highly sensitive identification of bacterial diarrheal pathogens. PMID:23884998

Lindsay, Brianna; Pop, Mihai; Antonio, Martin; Walker, Alan W; Mai, Volker; Ahmed, Dilruba; Oundo, Joseph; Tamboura, Boubou; Panchalingam, Sandra; Levine, Myron M; Kotloff, Karen; Li, Shan; Magder, Laurence S; Paulson, Joseph N; Liu, Bo; Ikumapayi, Usman; Ebruke, Chinelo; Dione, Michel; Adeyemi, Mitchell; Rance, Richard; Stares, Mark D; Ukhanova, Maria; Barnes, Bret; Lewis, Ian; Ahmed, Firoz; Alam, Meer Taifur; Amin, Ruhul; Siddiqui, Sabbir; Ochieng, John B; Ouma, Emmanuel; Juma, Jane; Mailu, Eunice; Omore, Richard; O'Reilly, Ciara E; Hannis, James; Manalili, Sheri; Deleon, Jonna; Yasuda, Irene; Blyn, Lawrence; Ranken, Raymond; Li, Feng; Housley, Roberta; Ecker, David J; Hossain, M Anowar; Breiman, Robert F; Morris, J Glenn; McDaniel, Timothy K; Parkhill, Julian; Saha, Debasish; Sampath, Rangarajan; Stine, O Colin; Nataro, James P

2013-10-01

302

Survey of Culture, GoldenGate Assay, Universal Biosensor Assay, and 16S rRNA Gene Sequencing as Alternative Methods of Bacterial Pathogen Detection  

PubMed Central

Cultivation-based assays combined with PCR or enzyme-linked immunosorbent assay (ELISA)-based methods for finding virulence factors are standard methods for detecting bacterial pathogens in stools; however, with emerging molecular technologies, new methods have become available. The aim of this study was to compare four distinct detection technologies for the identification of pathogens in stools from children under 5 years of age in The Gambia, Mali, Kenya, and Bangladesh. The children were identified, using currently accepted clinical protocols, as either controls or cases with moderate to severe diarrhea. A total of 3,610 stool samples were tested by established clinical culture techniques: 3,179 DNA samples by the Universal Biosensor assay (Ibis Biosciences, Inc.), 1,466 DNA samples by the GoldenGate assay (Illumina), and 1,006 DNA samples by sequencing of 16S rRNA genes. Each method detected different proportions of samples testing positive for each of seven enteric pathogens, enteroaggregative Escherichia coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shigella spp., Campylobacter jejuni, Salmonella enterica, and Aeromonas spp. The comparisons among detection methods included the frequency of positive stool samples and kappa values for making pairwise comparisons. Overall, the standard culture methods detected Shigella spp., EPEC, ETEC, and EAEC in smaller proportions of the samples than either of the methods based on detection of the virulence genes from DNA in whole stools. The GoldenGate method revealed the greatest agreement with the other methods. The agreement among methods was higher in cases than in controls. The new molecular technologies have a high potential for highly sensitive identification of bacterial diarrheal pathogens. PMID:23884998

Pop, Mihai; Antonio, Martin; Walker, Alan W.; Mai, Volker; Ahmed, Dilruba; Oundo, Joseph; Tamboura, Boubou; Panchalingam, Sandra; Levine, Myron M.; Kotloff, Karen; Li, Shan; Magder, Laurence S.; Paulson, Joseph N.; Liu, Bo; Ikumapayi, Usman; Ebruke, Chinelo; Dione, Michel; Adeyemi, Mitchell; Rance, Richard; Stares, Mark D.; Ukhanova, Maria; Barnes, Bret; Lewis, Ian; Ahmed, Firoz; Alam, Meer Taifur; Amin, Ruhul; Siddiqui, Sabbir; Ochieng, John B.; Ouma, Emmanuel; Juma, Jane; Mailu, Eunice; Omore, Richard; O'Reilly, Ciara E.; Hannis, James; Manalili, Sheri; DeLeon, Jonna; Yasuda, Irene; Blyn, Lawrence; Ranken, Raymond; Li, Feng; Housley, Roberta; Ecker, David J.; Hossain, M. Anowar; Breiman, Robert F.; Morris, J. Glenn; McDaniel, Timothy K.; Parkhill, Julian; Saha, Debasish; Sampath, Rangarajan; Stine, O. Colin; Nataro, James P.

2013-01-01

303

Pipette-friendly laminar flow patterning for cell-based assays†  

PubMed Central

Laminar flow patterning (LFP) is a characteristic method of microfluidic systems that allows two (or more) different solutions to flow side-by-side in a channel without convective mixing. This fluid behavior can be used to pattern cell suspensions, particles, and treatments as well as to create chemical gradients. LFP is typically implemented using syringe pumps and, for this reason, is most effective in constant flow scenarios such as long-term gradient generation. However, the complexity of using syringe pumps for patterning cell suspensions typically makes it a less attractive option than other standard patterning methods. We present a passive microfluidic method that enables short-term LFP of multiple fluids using a single pipette and allows each sample to be loaded in any sequence, at any point in time relative to one another. The proposed method reduces the complexity of LFP to be on a similar level as other cell patterning methods and is advantageous for cell-based applications such as co-culture and wound healing assays that require maximal exchange of soluble factors. PMID:21523269

Berthier, Erwin; Warrick, Jay; Casavant, Ben; Beebe, David J.

2012-01-01

304

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

PubMed

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

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

2006-01-01

305

Role of water-soluble matrix fraction, extracted from the nacre of Pinctada maxima, in the regulation of cell activity in abalone mantle cell culture ( Haliotis tuberculata)  

Microsoft Academic Search

In mollusks, the mantle is responsible for the secretion of an organic matrix that mineralizes to form the shell. A model of mantle cell culture has been established from the nacreous gastropod Haliotis tuberculata. First, viability of cells, quantified by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reduction assay, was monitored in order to determine a cell density and a time-culturing period

D. Sud; D. Doumenc; E. Lopez; C. Milet

2001-01-01

306

Effects of selenomethionine on cell growth and on S-adenosylmethionine metabolism in cultured malignant cells.  

PubMed Central

The effects of selenomethionine (SeMet) on the growth of 17 cultured cell lines were studied. SeMet in the culture medium of three hepatoma cell lines promoted cell growth at subcytotoxic levels (1-20 microM), but the growth of malignant lymphoid and myeloid cells was not stimulated. L-SeMet was cytotoxic to all 17 cell lines when assayed after culture for 3-10 days. A 50% growth inhibition was observed by 30-160 microM-SeMet in a culture medium containing 100 microM-methionine. SeMet cytotoxicity to normal (fibroblasts) and malignant cells was rather similar, excluding specific antineoplastic cytotoxicity. Cytotoxicity was increased by decreasing concentrations of methionine. The DL form of SeMet was less cytotoxic than the L form. L-SeMet was metabolized to a selenium analogue of S-adenosylmethionine approximately as effectively as the natural sulphur analogue methionine in malignant R1.1 lymphoblasts. Concomitantly, S-adenosylmethionine pools were decreased. This occurred early and at cytotoxic SeMet levels. Methionine adenosyltransferase activity was not altered by SeMet treatment. ATP pools were not affected early, and decreases in the synthesis of DNA and protein took place late and were apparently related to cell death. RNA synthesis was slightly stimulated at low cytotoxic SeMet levels by 24 h, but was markedly inhibited after 48 h. The SeMet analogue of S-adenosylmethionine could be effectively utilized in a specific enzymic transmethylation. Neither S-adenosylhomocysteine nor its selenium analogue accumulated in the treated cells. These findings together suggest a direct or indirect involvement of S-adenosylmethionine metabolism in SeMet cytotoxicity, but exclude a gross blockage of transmethylations. PMID:2339986

Kajander, E O; Harvima, R J; Kauppinen, L; Akerman, K K; Martikainen, H; Pajula, R L; Karenlampi, S O

1990-01-01

307

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

308

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

309

Isolation and culture of larval cells from C. elegans.  

PubMed

Cell culture is an essential tool to study cell function. In C. elegans the ability to isolate and culture cells has been limited to embryonically derived cells. However, cells or blastomeres isolated from mixed stage embryos terminally differentiate within 24 hours of culture, thus precluding post-embryonic stage cell culture. We have developed an efficient and technically simple method for large-scale isolation and primary culture of larval-stage cells. We have optimized the treatment to maximize cell number and minimize cell death for each of the four larval stages. We obtained up to 7.8×10(4) cells per microliter of packed larvae, and up to 97% of adherent cells isolated by this method were viable for at least 16 hours. Cultured larval cells showed stage-specific increases in both cell size and multinuclearity and expressed lineage- and cell type-specific reporters. The majority (81%) of larval cells isolated by our method were muscle cells that exhibited stage-specific phenotypes. L1 muscle cells developed 1 to 2 wide cytoplasmic processes, while L4 muscle cells developed 4 to 14 processes of various thicknesses. L4 muscle cells developed bands of myosin heavy chain A thick filaments at the cell center and spontaneously contracted ex vivo. Neurons constituted less than 10% of the isolated cells and the majority of neurons developed one or more long, microtubule-rich protrusions that terminated in actin-rich growth cones. In addition to cells such as muscle and neuron that are high abundance in vivo, we were also able to isolate M-lineage cells that constitute less than 0.2% of cells in vivo. Our novel method of cell isolation extends C. elegans cell culture to larval developmental stages, and allows use of the wealth of cell culture tools, such as cell sorting, electrophysiology, co-culture, and high-resolution imaging of subcellular dynamics, in investigation of post-embryonic development and physiology. PMID:21559335

Zhang, Sihui; Banerjee, Diya; Kuhn, Jeffrey R

2011-01-01

310

An assay for serum cytotoxicity against erythroid precursor cells in pure red cell aplasia.  

PubMed

Several reports have indicated that a circulating serum inhibitor (antibody) is involved in the pathogenesis of acquired pure red cell aplasia (PRCA). In the present study, the pathophysiologic significance of this inhibitor was assessed according to the status of erythroid progenitor cells in the bone marrow. So far, direct proof for the antibody acting against erythroid stemcells was lacking. Employing an "in vitro" assay, erythroid colony forming cell (CFU-e) numbers in PRCA marrow were quantified and the cytotoxic effect of PRCA serum on CFU-e was investigated. It was revealed that the CFU-e population size in the marrow of PRCA patients was severely reduced; at the same time the relative number of myeloid colony forming cells was normal. The serum was demonstrated to contain a factor cell which was cytotoxic to CFU-e, in the presence of complement. The results indicate that inhibition of erythropoiesis in PRCA is achieved by a complement dependent plasma factor which eliminates or inactivates CFU-e and which constitutes an effective block at the precursor cell level in the differentiation pathway of the erythroid line. The data present a practical assay for measuring cytotoxic factors affecting erythroid stem cells. PMID:597564

Löwenberg, B; Ghio, R

1977-11-01

311

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

Microsoft Academic Search

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

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

2011-01-01

312

A high throughput electrochemiluminescent cell-binding assay for therapeutic anti-CD20 antibody selection.  

PubMed

A cell-based ELISA using suspension WIL2 cells in 96-well format was previously developed for measuring relative binding affinities of humanized anti-CD20 variants. We further developed a new cell-binding assay that uses high binding capacity carbon electrode plates for rapid attachment of suspension WIL2 cells and electrochemiluminescence for detection. Compared to the cell-based ELISA, which requires centrifugation for the manual wash steps, significant improvement in assay throughput was achieved by using a microplate washer. The assay can be performed on both 96- and 384-well plates with a standard curve range of 2.74-2000 ng/ml, which is wider than the range of 15.6-1000 ng/ml for the cell-based ELISA. Using CD20 expressing CHO cell clones, surface expression of >or=33,000 CD20 molecules was sufficient to obtain a dose-response curve in 384-well format. Relative affinities of 15 humanized variants correlated well (r(2)=0.94) between electrochemiluminescent cell-binding assay and cell-based ELISA. A competitive assay format, using mouse anti-CD20 antibody as the tracer, with a dose-response range of 27.4-20,000 ng/ml was also developed. The new cell-binding assay method can be used to efficiently support humanization process for selection of anti-CD20 antibody drug candidates and to characterize antibody binding to other cell surface proteins. PMID:16814318

Lu, Yanmei; Wong, Wai Lee; Meng, Y Gloria

2006-07-31

313

Assay for trans-p-coumaroyl esterase using a specific substrate from plant cell walls  

SciTech Connect

Cell walls of Coastal Bermuda grass (Cynodon dactylon) were treated with polysaccharide hydrolases to release O-(5-O-(trans-p-coumaroyl)-{alpha}-L-arabinofuranosyl)-(1{yields}3)-O-{beta}-D-xylopyranosyl-(1{yields}4)-D-xylopyranose (PAXX) which was isolated by liquid chromatography. The isolated PAXX was greater than 95% pure as determined by H NMR and was used as substrate for a sensitive assay of trans-p-coumaroyl esterase. PAXX was hydrolyzed by culture filtrates from the anaerobic fungus Neocallimastix MC-2. The trans-p-coumaric acid released by enzymatic hydrolysis was assayed by reverse-phase HPLC, and as little as 100 ng of acid could be determined. Steady-state velocities for the release of the acid obeyed Michaelis-Menten kinetics. V{sub max} was determined to be 1.17 {mu}mol min{sup {minus}1} mg{sup {minus}1} and K{sub m} 13.2 {mu}m at pH 7.5 and 30C.

Borneman, W.S. (Dept. of Agriculture, Athens, GA (United States) Univ. of Georgia, Athens (United States)); Hartley, R.D.; Himmelsbach, D.S. (Dept. of Agriculture, Athens, GA (United States)); Ljungdahl, L.G. (Univ. of Georgia, Athens (United States))

1990-01-01

314

Assay for trans-p-coumaroyl esterase using a specific substrate from plant cell walls.  

PubMed

Cell walls of Coastal Bermuda grass (Cynodon dactylon) were treated with polysaccharide hydrolases to release O-[5-O-(trans-p-coumaroyl)-alpha-L-arabinofuranosyl]-(1----3)-O-be ta-D- xylopyranosyl-(1----4)-D-xylopyranose (PAXX) which was isolated by liquid chromatography. The isolated PAXX was greater than 95% pure as determined by 1H NMR and was used as substrate for a sensitive assay of trans-p-coumaroyl esterase. PAXX was hydrolyzed by culture filtrates from the anaerobic fungus Neocallimastix MC-2. The trans-p-coumaric acid released by enzymatic hydrolysis was assayed by reverse-phase HPLC, and as little as 100 ng of acid could be determined. Steady-state velocities for the release of the acid obeyed Michaelis-Menten kinetics. Vmax was determined to be 1.17 mumol min-1 mg-1 and Km 13.2 microM at pH 7.5 and 30 degrees C. PMID:2285139

Borneman, W S; Hartley, R D; Himmelsbach, D S; Ljungdahl, L G

1990-10-01

315

Development of a Lentivirus Vector-Based Assay for Non-Destructive Monitoring of Cell Fusion Activity  

PubMed Central

Cell-to-cell fusion can be quantified by endowing acceptor and donor cells with latent reporter genes/proteins and activators of these genes/proteins, respectively. One way to accomplish this goal is by using a bipartite lentivirus vector (LV)-based cell fusion assay system in which the cellular fusion partners are transduced with a flippase-activatable Photinus pyralis luciferase (PpLuc) expression unit (acceptor cells) or with a recombinant gene encoding FLPeNLS+, a nuclear-targeted and molecularly evolved version of flippase (donor cells). Fusion of both cell populations will lead to the FLPe-dependent generation of a functional PpLuc gene. PpLuc activity is typically measured in cell lysates, precluding consecutive analysis of one cell culture. Therefore, in this study the PpLuc-coding sequence was replaced by that of Gaussia princeps luciferase (GpLuc), a secretory protein allowing repeated analysis of the same cell culture. In myotubes the spread of FLPeNLS+ may be limited due to its nuclear localization signal (NLS) causing low signal outputs. To test this hypothesis, myoblasts were transduced with LVs encoding either FLPeNLS+ or an NLS-less version of FLPe (FLPeNLS?) and subsequently co-cultured in different ratios with myoblasts containing the FLPe-activatable GpLuc expression cassette. At different times after induction of cell-to-cell fusion the GpLuc activity in the culture medium was determined. FLPeNLS+ and FLPeNLS? both activated the latent GpLuc gene but when the percentage of FLPe-expressing myoblasts was limiting, FLPeNLS+ generally yielded slightly higher signals than FLPeNLS? while at low acceptor-to-donor cell ratios FLPeNLS? was usually superior. The ability of FLPeNLS+ to spread through myofibers and to induce reporter gene expression is thus not limited by its NLS. However, at high FLPe concentrations the presence of the NLS negatively affected reporter gene expression. In summary, a rapid and simple chemiluminescence assay for quantifying cell-to-cell fusion progression based on GpLuc has been developed. PMID:25028973

Neshati, Zeinab; Liu, Jia; Zhou, Guangqian; Schalij, Martin J.; de Vries, Antoine A. F.

2014-01-01

316

A simple and rapid method to assay triacylglycerol in cells and tissues.  

PubMed

We have developed a reliable, rapid, and economical assay for the quantification of triacylglycerol (TG) in cells and animal tissues. In a few hours, this assay quantifies microgram amounts of TG from tens or even hundreds of samples. The protocol includes an organic extraction to partition TG away from proteins and other hydrophilic molecules found in cells and tissues that may interfere with the colorimetric enzyme-linked TG detection method. In addition, this assay is economical, as no expensive reagents, supplies, or equipment are needed. Another benefit of this assay is that it does not require environmentally unfriendly halogenated solvents. PMID:17717377

Schwartz, Danielle M; Wolins, Nathan E

2007-11-01

317

Culturing conditions affecting the production of anthocyanin in suspended cell cultures of strawberry  

Microsoft Academic Search

The increase of anthocyanin content in suspended cell cultures of strawberry varied with the increase in the amount of pigmentation in pigmented cells and in number of pigmented cells in a culture. The anthocyanin yield was enhanced by increasing light irradiation, and this may have resulted from increased accumulation of anthocyanin in pigmented cells. The increased anthocyanin yield for the

Kenji Sato; Mamoru Nakayama; Jun-ichi Shigeta

1996-01-01

318

Evaluation of 309 Environmental Chemicals Using a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity Assay  

PubMed Central

The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES) cells provide an in vitro model of embryonic development and an alternative method for assessing developmental toxicity. Here, we evaluated 309 environmental chemicals, mostly food-use pesticides, from the ToxCast™ chemical library using a mouse ES cell platform. ES cells were cultured in the absence of pluripotency factors to promote spontaneous differentiation and in the presence of DMSO-solubilized chemicals at different concentrations to test the effects of exposure on differentiation and cytotoxicity. Cardiomyocyte differentiation (?,? myosin heavy chain; MYH6/MYH7) and cytotoxicity (DRAQ5™/Sapphire700™) were measured by In-Cell Western™ analysis. Half-maximal activity concentration (AC50) values for differentiation and cytotoxicity endpoints were determined, with 18% of the chemical library showing significant activity on either endpoint. Mining these effects against the ToxCast Phase I assays (?500) revealed significant associations for a subset of chemicals (26) that perturbed transcription-based activities and impaired ES cell differentiation. Increased transcriptional activity of several critical developmental genes including BMPR2, PAX6 and OCT1 were strongly associated with decreased ES cell differentiation. Multiple genes involved in reactive oxygen species signaling pathways (NRF2, ABCG2, GSTA2, HIF1A) were strongly associated with decreased ES cell differentiation as well. A multivariate model built from these data revealed alterations in ABCG2 transporter was a strong predictor of impaired ES cell differentiation. Taken together, these results provide an initial characterization of metabolic and regulatory pathways by which some environmental chemicals may act to disrupt ES cell growth and differentiation. PMID:21666745

Chandler, Kelly J.; Barrier, Marianne; Jeffay, Susan; Nichols, Harriette P.; Kleinstreuer, Nicole C.; Singh, Amar V.; Reif, David M.; Sipes, Nisha S.; Judson, Richard S.; Dix, David J.; Kavlock, Robert; Hunter, Edward S.; Knudsen, Thomas B.

2011-01-01

319

Implementing CompacT SelecT Automated Cell Culture in Support of Alzheimer's Disease-Targeted Drug Discovery  

Microsoft Academic Search

The CompacT SelecT is the latest generation automated cell culture system in the SelecT product line allowing incubation of up to 90 T-175 flasks and preparation of 210 assay-ready plates. We have successfully implemented the CompacT SelecT in support of a number of cell-based assays used in our Alzheimer's disease (AD) lead optimization programs. One of the distinguishing features of

Zhuangwei Lou; Yiqun Wang; Stan Nawoschik; David Riddell; Robert Martone; John Dunlop

2008-01-01

320

Rotating bio-reactor cell culture apparatus  

NASA Technical Reports Server (NTRS)

A bioreactor system is described in which a tubular housing contains an internal circularly disposed set of blade members and a central tubular filter all mounted for rotation about a common horizontal axis and each having independent rotational support and rotational drive mechanisms. The housing, blade members and filter preferably are driven at a constant slow speed for placing a fluid culture medium with discrete microbeads and cell cultures in a discrete spatial suspension in the housing. Replacement fluid medium is symmetrically input and fluid medium is symmetrically output from the housing where the input and the output are part of a loop providing a constant or intermittent flow of fluid medium in a closed loop.

Schwarz, Ray P. (inventor); Wolf, David A. (inventor)

1991-01-01

321

Differentiated cultures of primary hamster tracheal airway epithelial cells  

Microsoft Academic Search

Summary  Primary airway epithelial cell cultures can provide a faithful representation of the in vivo airway while allowing for a controlled\\u000a nutrient source and isolation from other tissues or immune cells. The methods used have significant differences based on tissue\\u000a source, cell isolation, culture conditions, and assessment of culture purity. We modified and optimized a method for generating\\u000a tracheal epithelial cultures

Regina K. Rowe; Steven L. Brody; Andrew Pekosz

2004-01-01

322

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; Lusebrink, Jessica; Pieper, Monika; Tillmann, Ramona L.; Brockmann, Michael

2014-01-01

323

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

324

Development and evaluation of an anchorage-independent agar-based clonal assay for human primary breast carcinoma cells  

SciTech Connect

The development and evaluation of an anchorage-independent clonal cytotoxic assay for primary human breast carcinoma cells is described in this thesis. This assay was developed in three stages which include: (1) the optimization of the production of a monodispersed cell suspension from solid breast carcinomas, (2) the systematic development of a growth medium for the clonal growth of these cells, and (3) the adaptation of these methods for use in the quantitation of cytotoxicity. The results of these studies indicated that hydrocortisone, fetal bovine serum and red blood cells stimulated the clonal growth of breast carcinoma cells. The optimal concentrations of these three factors were simultaneously determined using response surface methodology. These culture conditions were then used to develop radiation-cytotoxicity assays for both primary and recurrent breast carcinomas. The methodology developed and evaluated in this thesis may be useful to: (1) study the biology and radiobiology of human breast cancer, (2) customize the treatment of individual breast cancer patients, and (3) identify and/or develop new drugs and/or other treatment modalities for breast cancer.

Besch, G.J.

1985-01-01

325

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

326

Effects of dexamethasone on human lens epithelial cells in culture  

PubMed Central

Purpose Treatment with glucocorticoids is a well known risk factor for cataract development, although the pathogenic mechanism has not been elucidated. The aim of the study was to investigate the effects of glucocorticoids in cultured human lens epithelial cells. Methods Human lens epithelial cells (HLECs) were exposed to dexamethasone for 24 h. The number of viable cells was determined using the 3-[4, 5-dimethylthiazolyl-2]-2, 5-diphenyltetrazolium bromide (MTT) assay, and proliferation was quantified using Ki-67. Apoptosis was investigated by measuring caspase-3 activity and by evaluating nuclear morphology of cells stained with Hoechst 33342. Mitochondria depolarization was measured using the potential-sensitive color, JC-1. Cells were assayed for changes in superoxide production using dihydroethidium (HET), for alterations in peroxide production using dichlorofluorescein diacetate (DCFH-DA), and for glutathione (GSH) variations using monochlorobimane (MCB). Caspase-3 activity was also measured in HLECs simultaneously exposed to dexamethasone and the glucocorticoid antagonist, RU486. Results Low doses of dexamethasone (0.1 µM) resulted in increased proliferation of HLECs. Apoptosis was increased in HLECs exposed to 1 µM, 10 µM, and 100 µM of dexamethasone as revealed by nuclear morphology studies. Apoptosis was also confirmed by measuring caspase-3 activation. No effect on superoxide production by dexamethasone was seen. There were no effects on GSH levels or mitochondrial depolarization either. Only the highest concentration of dexamethasone (100 µM) caused an increase in peroxide production. In HLECs incubated with the glucocorticoid antagonist, RU486, apoptosis was induced at a lower concentration of dexamethasone (0.1 µM) than with dexamethasone alone. Conclusions Low doses of dexamethasone cause a moderate increase in proliferation of cultured HLECs. Slightly higher but still physiologically relevant concentrations of dexamethasone result in a dose-dependent increase in apoptosis. Dexamethasone-induced apoptosis in HLECs does not seem to involve oxidative mechanisms. The proapoptotic effect of dexamethasone does not appear to act through the glucocorticoid receptor. Effects on proliferation and/or dysregulation of apoptosis in lens epithelial cells may be an important factor in human steroid-induced posterior subcapsular cataract. PMID:18648526

Carlsson, T.; Karlsson, J-O.; Jonhede, S.; Zetterberg, M.

2008-01-01

327

Effects of perfluorocarbon emulsions on cultured human endothelial cells.  

PubMed

Perfluorocarbons (PFCs) and their emulsions (PFCEs) were used in organ preservation before transplantation, but not in organ perfusion. Our purpose was to achieve organ perfusion with a PFCE at room temperature or at 37 degrees C, i. e. with oxygenation, to prevent damages related to reoxygenation after hypoxia. Therefore, we first investigated the effect of such emulsions on endothelial cells, the first cells to be in contact with the emulsion. A stem emulsion was prepared from perfluorooctyl bromide (90% w/v), emulsified with egg yolk phospholipids (2% w/v) and stabilized with a mixed fluorocarbon-hydrocarbon "molecular dowel" (1.4% w/v) (droplets of ca 0.2 micron in diameter). This emulsion was found to be stable when diluted with cell culture media or organ preservation fluids. Endothelial cells from human umbilical vein (HUVECs) were cultured in multiwell plates in M199 medium (with growth factors, 10% foetal calf serum and 5% human serum). Confluent cells were incubated overnight with 51Cr, washed and overlayed with M199 (control) or the above PFCE diluted 2x or 4x with M199 (test). After incubation, the cytotoxicity of the PFCEs was estimated by measuring 51Cr release and observing cell morphology by electron and light microscopy. The percentages of released 51Cr were identical to those of the control cells for the 2x, 3x or 4x diluted PFCEs at 4, 25 or 37 degrees C. After return to the M199 medium, the cells grew and multiplied normally. We conclude that the diluted PFCEs were devoid of cytotoxicity. The 2x diluted PFCE was however partially taken up by the cells: by microscopy, we observed intracellular PFC droplets and by density gradient analysis we found a slight increase in cellular density. The diluted PFCEs were compared to classical organ preservation solutions : HUVECs were incubated with UW (University of Wisconsin) or EC (EuroCollins) solutions at +4 and 37 degrees C (3, 17 or 24 h of incubation). The solutions were observed to be toxic to the cells under these conditions, with cell mortality after return to the M199 medium. This cytotoxicity may be attributed to the high K+ concentration of UW and EC, since similar assays performed on HUVECs with Hank's solution adjusted to 100 mM K+ showed a similar % of 51Cr release. UW and EC are therefore not acceptable as dilution media for PFCEs. PMID:9352061

Mathy-Hartert, M; Krafft, M P; Deby, C; Deby-Dupont, G; Meurisse, M; Lamy, M; Riess, J G

1997-11-01

328

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

329

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

NASA Astrophysics Data System (ADS)

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

Hondroulis, Evangelia; Liu, Chang; Li, Chen-Zhong

2010-08-01

330

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

331

Adult human aortic cells in primary culture: heterogeneity in shape  

Microsoft Academic Search

Summary Adult human aortic cells have different shapes in situ. To determine whether populations of cultured aortic cells are also polymorphic, a technique for separation of cells from the intimal and medial layers of the human aorta by enzymatic dispersion of the vascular tissue was employed. It was established that aortic cells are polymorphic in primary culture, at least within

Alexander N. Orekhov; Anatoly V. Krushinsky; Elena R. Andreeva; Vadim S. Repin; Vladimir N. Smirnov

1986-01-01

332

Differentiated cultures of primary hamster tracheal airway epithelial cells.  

PubMed

Primary airway epithelial cell cultures can provide a faithful representation of the in vivo airway while allowing for a controlled nutrient source and isolation from other tissues or immune cells. The methods used have significant differences based on tissue source, cell isolation, culture conditions, and assessment of culture purity. We modified and optimized a method for generating tracheal epithelial cultures from Syrian golden hamsters and characterized the cultures for cell composition and function. Soon after initial plating, the epithelial cells reached a high transepithelial resistance and formed tight junctions. The cells differentiated into a heterogeneous, multicellular culture containing ciliated, secretory, and basal cells after culture at an air-liquid interface (ALI). The secretory cell populations initially consisted of MUC5AC-positive goblet cells and MUC5AC/CCSP double-positive cells, but the makeup changed to predominantly Clara cell secretory protein (CCSP)-positive Clara cells after 14 d. The ciliated cell populations differentiated rapidly after ALI, as judged by the appearance of beta tubulin IV-positive cells. The cultures produced mucus, CCSP, and trypsin-like proteases and were capable of wound repair as judged by increased expression of matrilysin. Our method provides an efficient, high-yield protocol for producing differentiated hamster tracheal epithelial cells that can be used for a variety of in vitro studies including tracheal cell differentiation, airway disease mechanisms, and pathogen-host interactions. PMID:15780007

Rowe, Regina K; Brody, Steven L; Pekosz, Andrew

2004-01-01

333

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

334

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

335

A novel asymmetric 3D in-vitro assay for the study of tumor cell invasion  

Microsoft Academic Search

BACKGROUND: The induction of tumor cell invasion is an important step in tumor progression. Due to the cost and slowness of in-vivo invasion assays, there is need for quantitative in-vitro invasion assays that mimic as closely as possible the tumor environment and in which conditions can be rigorously controlled. METHODS: We have established a novel asymmetric 3D in-vitro invasion assay

Vera Brekhman; Gera Neufeld

2009-01-01

336

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 nonmyocyte 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 that 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 cultured in HARVs 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 HARVs 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 tissuelike organization of cardiac cells in vitro.

Akins, R. E.; Schroedl, N. A.; Gonda, S. R.; Hartzell, C. R.

1997-01-01

337

The antioxidant effect of the Malaysian Gelam honey on pancreatic hamster cells cultured under hyperglycemic conditions.  

PubMed

Type 2 diabetes consists of progressive hyperglycemia, insulin resistance, and pancreatic ?-cell failure which could result from glucose toxicity, inflammatory cytokines, and oxidative stress. In the present study, we investigate the effect of pretreatment with Gelam honey (Melaleuca spp.) and the individual flavonoid components chrysin, luteolin, and quercetin, on the production of reactive oxygen species (ROS), cell viability, lipid peroxidation, and insulin content in hamster pancreatic cells (HIT-T15 cells), cultured under normal and hyperglycemic conditions. Phenolic extracts from a local Malaysian species of Gelam honey (Melaleuca spp.) were prepared using the standard extraction methods. HIT-T15 cells were cultured in 5 % CO2 and then preincubated with Gelam honey extracts (20, 40, 60, and 80 ?g/ml) as well as some of its flavonoid components chrysin, luteolin, and quercetin (20, 40, 60, and 80 ?M), prior to stimulation by 20 and 50 mM of glucose. The antioxidative effects were measured in these cultured cells at different concentrations and time point by DCFH-DA assay. Pretreatment of cells with Gelam honey extract or the flavonoid components prior to culturing in 20 or 50 mM glucose showed a significant decrease in the production of ROS, glucose-induced lipid peroxidation, and a significant increase in insulin content and the viability of cells cultured under hyperglycemic condition. Our results show the in vitro antioxidative property of the Gelam honey and the flavonoids on the ?-cells from hamsters and its cytoprotective effect against hyperglycemia. PMID:23584372

Batumalaie, Kalaivani; Qvist, Rajes; Yusof, Kamaruddin Mohd; Ismail, Ikram Shah; Sekaran, Shamala Devi

2014-05-01

338

Salt tolerance in cultured cells of Spartina pectinata  

Microsoft Academic Search

Suspension cultures with cell doubling times of ca. 2 days were developed from the halophytic grass Spartina pectinata. Maximum rates of exponential growth measured by direct cell counts and by total culture packed-cell-volume were not significantly reduced by NaCl up to 200 mM but dropped beyond this point. In contrast, total cell production over a one week culture cycle, by

R. Scott Warren; Lisa M. Baird; Angela K. Thompson

1985-01-01

339

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 virtually infected or stably transformed insect cells containing a gene encoding the described polypeptide. The insect cells can also be a host for viral production.

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

1997-01-01

340

Characterization of zinc-induced neuronal death in primary cultures of rat cerebellar granule cells.  

PubMed

Although zinc is essential for the activity of numerous biological systems, and zinc deficiency has been associated with various pathologies, this metal can also exert direct neurotoxic action. In primary cultures of rat cerebellar granule neurons, a brief, 15- to 30-min exposure to zinc (100-500 microM) resulted in concentration-dependent delayed neuronal death. The toxicity of zinc depended on the maturity of the neuronal cultures-it was not apparent prior to Day 5 and it reached a plateau at about 9-10 days in vitro. We assayed cell injury by measuring mitochondrial functioning (MTT assay) and cell death with the trypan blue exclusion assay. Apoptosis was assayed by the morphological appearance of cells following fluorescence staining with propidium iodide and by the in situ TUNEL technique. Mitochondrial injury was an early result of zinc treatment. Actinomycin D, an inhibitor of macromolecular synthesis, attenuated delayed cell death. The calcium channel blockers nimodipine and amlodipine reduced both mitochondrial injury and cell death; the blockade of ionotropic glutamate receptors with MK-801 or CNQX was ineffective. These results suggest that calcium channel-blocker-sensitive mitochondrial injury and DNA damage are operative in the protein-synthesis-dependent neurotoxicity of zinc. An imbalance of zinc homeostasis might play a role in the pathophysiology of apoptosis-associated neurodegenerative disorders. PMID:9225750

Manev, H; Kharlamov, E; Uz, T; Mason, R P; Cagnoli, C M

1997-07-01

341

Assessment of cell death studies by monitoring hydrogen peroxide in cell culture.  

PubMed

Hydrogen peroxide (H2O2) has been widely used to study the oxidative stress response. However, H2O2 is unstable and easily decomposes into H2O and O2. Consequently, a wide range of exposure times and treatment concentrations has been described in the literature. In the present study, we established a ferrous oxidation-xylenol orange (FOX) assay, which was originally described for food and body liquids, as a method for the precise quantification of H2O2 concentrations in cell culture media. We observed that the presence of FCS and high cell densities significantly accelerate the decomposition of H2O2, therefore acting as a protection against cell death by accidental necrosis. PMID:24747006

Hirsch, Irina; Prell, Erik; Weiwad, Matthias

2014-07-01

342

Problems utilizing an enzyme sensitive site assay for photorepair of exogenous DNA with cell-free  

E-print Network

-B (UVB) damage repair ability of seven Ontario amphibian spe- cies using an enzyme sensitive siteNOTE Problems utilizing an enzyme sensitive site assay for photorepair of exogenous DNA with cell-free extracts made from amphibian embryos M. Alex Smith Abstract: Attempts were made to assay the ultraviolet

Smith, M. Alex

343

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

344

Escherichia coli Enterotoxin-Induced Steroidogenesis in Cultured Adrenal Tumor Cells  

PubMed Central

A heat-labile, Pronase-sensitive factor has been partially purified from cell-free culture filtrates of enterotoxigenic Escherichia coli. The partially purified factor contains both protein and carbohydrate moieties and appears to be E. coli enterotoxin (ECT). ECT binds to cultured adrenal tumor cells rapidly and irreversibly leads to adenosine 3?, 5?-cyclic monophosphate formation and steroidogenesis after a 60-min lag phase. Further studies indicate that it interacts with the cholera toxin receptor site on adrenal cells rather than the adrenocorticotropin receptor to activate adenyl cyclase. Mixed gangliosides block stimulation of steroidogenesis in response to both E. coli and cholera enterotoxin. In contrast to adrenocorticotropin, ECT has no additive effect on cholera toxin-induced steroidogenesis. The protein moiety of ECT is similar to cholera enterotoxin because horse serum anticholeragenoid prevented stimulation of steroidogenesis by either enterotoxin. Cultured adrenal cells provide a quantitative assay system that has facilitated the purification and characterization of E. coli enterotoxin. Images PMID:4366919

Kwan, Catherine N.; Wishnow, Rodney M.

1974-01-01

345

Waste-water assay with continuous algal cultures: The effect of mercuric acetate on the growth of some marine dinoflagellates  

Microsoft Academic Search

The effect of mercuric acetate was studied in culture experiments with the dinoflagellates Scrippsiella faeroense (Paulsen) Balech et Soares, Prorocentrum micans Ehrenberg and Gymnodinium splendens Lebour. Impairment of growth rates, in vivo chlorophyll fluorescence, maximum cell densities and morphological changes served as criteria for assessing sublethal influences. Tests were made using the batch- and continuous-culture techniques. Addition of Hg at

H. Kayser

1976-01-01

346

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

347

Failure to Detect Chlamydia pneumoniae by Cell Culture and Polymerase Chain Reaction in Major Arteries of 93 Patients with Atherosclerosis  

Microsoft Academic Search

  \\u000a To detect Chlamydia pneumoniae in punch specimens of the aortic wall of 61 patients undergoing coronary-aortic bypass graft, and carotid atheromas of 32\\u000a patients undergoing carotid endarterectomy, cell culture (HEp-2 cells) and two polymerase chain reaction assays in two different\\u000a laboratories were used. All cultures and polymerase chain reaction tests for Chlamydia pneumoniae were negative. Further studies are required

J. Bishara; S. Pitlik; A. Kazakov; G. Sahar; M. Haddad; A. Vojdani; S. Rosenberg; Z. Samra

2003-01-01

348

Gravity, chromosomes, and organized development in aseptically cultured plant cells  

NASA Technical Reports Server (NTRS)

The objectives of the PCR experiment are: to test the hypothesis that microgravity will in fact affect the pattern and developmental progression of embryogenically competent plant cells from one well-defined, critical stage to another; to determine the effects of microgravity in growth and differentiation of embryogenic carrot cells grown in cell culture; to determine whether microgravity or the space environment fosters an instability of the differentiated state; and to determine whether mitosis and chromosome behavior are adversely affected by microgravity. The methods employed will consist of the following: special embryogenically competent carrot cell cultures will be grown in cell culture chambers provided by NASDA; four cell culture chambers will be used to grow cells in liquid medium; two dishes (plant cell culture dishes) will be used to grow cells on a semi-solid agar support; progression to later embryonic stages will be induced in space via crew intervention and by media manipulation in the case of liquid grown cell cultures; progression to later stages in case of semi-solid cultures will not need crew intervention; embryo stages will be fixed at a specific interval (day 6) in flight only in the case of liquid-grown cultures; and some living cells and somatic embryos will be returned for continued post-flight development and 'grown-out.' These will derive from the semi-solid grown cultures.

Krikorian, Abraham D.

1993-01-01

349

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

350

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

351

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

352

In vitro culture of C. elegans somatic cells.  

PubMed

Because of technical hurdles, large-scale cell culture methods have not been widely exploited until recently for the study of Caenorhabditis elegans. Culturing differentiated cells from larvae and adult worms is probably not technically feasible because of difficulties in removing the animal's cuticle and dissociating cells. In contrast, large numbers of developing embryo cells can be isolated relatively easily. When placed in culture, embryo cells undergo terminal differentiation within 24 h. Cultured embryo cells have been used recently to characterize ion channel function and regulation and to determine cell specific gene expression patterns. This chapter will provide a detailed description of the methods for isolating and culturing C. elegans embryo cells. PMID:16988440

Strange, Kevin; Morrison, Rebecca

2006-01-01

353

Genotoxicity of alkene epoxides in human peripheral blood mononuclear cells and HL60 leukaemia cells evaluated with the comet assay.  

PubMed

Volatile organic compounds (VOCs) exert their carcinogenic activity through the production of epoxide metabolites. Because of their high reactivity some epoxides are also produced in the chemical industry for the synthesis of other compounds. Therefore, human exposure to VOCs epoxides does occur and may be an important human health concern. In this study, the in vitro genotoxic potential of epoxides originating from 1,3-butadiene (3,4-epoxy-1-butene: EB; 1,2:3,4-diepoxybutane: DEB), isoprene (3,4-epoxy-2-methyl-1-butene: IO), styrene (styrene-7,8-oxide: SO), propylene (propylene oxide: PO) and 1-butene (1,2-epoxy-butane: BO) in human peripheral blood mononuclear cells (PBMCs) and promyelocytic leukaemia cells (HL60) was measured with the comet assay (single-cell gel electrophoresis, SCGE). The effect of inclusion of foetal calf serum (FCS, 5%) in the cell-culture medium and different durations of exposure (2h, 24h) were also investigated. All epoxides tested produced DNA damage in a concentration range that did not reduce cell viability. HL60 cells were more resistant than PBMCs to the DNA damage induced by the different epoxides. With the exception of IO, the treatment for 24h resulted in an increase of DNA damage. FCS slightly protected PBMCs from the genotoxic effects induced by IO and BO, whilst no such effect was noted for the other compounds. Overall, the dose-dependent effects that were seen allowed us to define a genotoxicity scale for the different epoxides as follows: SO>EB>DEB>IO>PO>BO, which is in partial agreement with the International Agency for Research on Cancer (IARC) classification of the carcinogenic hazards. PMID:22285587

Fabiani, Roberto; Rosignoli, Patrizia; De Bartolomeo, Angelo; Fuccelli, Raffaela; Morozzi, Guido

2012-08-30

354

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

355

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

356

Single Cell Adhesion Assay Using Computer Controlled Micropipette  

PubMed Central

Cell adhesion is a fundamental phenomenon vital for all multicellular organisms. Recognition of and adhesion to specific macromolecules is a crucial task of leukocytes to initiate the immune response. To gain statistically reliable information of cell adhesion, large numbers of cells should be measured. However, direct measurement of the adhesion force of single cells is still challenging and today’s techniques typically have an extremely low throughput (5–10 cells per day). Here, we introduce a computer controlled micropipette mounted onto a normal inverted microscope for probing single cell interactions with specific macromolecules. We calculated the estimated hydrodynamic lifting force acting on target cells by the numerical simulation of the flow at the micropipette tip. The adhesion force of surface attached cells could be accurately probed by repeating the pick-up process with increasing vacuum applied in the pipette positioned above the cell under investigation. Using the introduced methodology hundreds of cells adhered to specific macromolecules were measured one by one in a relatively short period of time (?30 min). We blocked nonspecific cell adhesion by the protein non-adhesive PLL-g-PEG polymer. We found that human primary monocytes are less adherent to fibrinogen than their in vitro differentiated descendants: macrophages and dendritic cells, the latter producing the highest average adhesion force. Validation of the here introduced method was achieved by the hydrostatic step-pressure micropipette manipulation technique. Additionally the result was reinforced in standard microfluidic shear stress channels. Nevertheless, automated micropipette gave higher sensitivity and less side-effect than the shear stress channel. Using our technique, the probed single cells can be easily picked up and further investigated by other techniques; a definite advantage of the computer controlled micropipette. Our experiments revealed the existence of a sub-population of strongly fibrinogen adherent cells appearing in macrophages and highly represented in dendritic cells, but not observed in monocytes. PMID:25343359

Salanki, Rita; Hos, Csaba; Orgovan, Norbert; Peter, Beatrix; Sandor, Noemi; Bajtay, Zsuzsa; Erdei, Anna; Horvath, Robert; Szabo, Balint

2014-01-01

357

Cell-based assay for the determination of temperature sensitive and cold adapted phenotypes of influenza viruses.  

PubMed

The determination of temperature sensitive (ts) and cold adapted (ca) phenotype for influenza A and B strains has been conducted traditionally using embryonated chicken eggs. As attempts are made to move away from the use of eggs in the manufacturing process of influenza vaccines, it will become useful to develop cell-based assays to support cell culture-based vaccine production. In this study, MDCK cells have been evaluated as a tool for determining the ts and ca phenotypes associated with live attenuated influenza viruses. Direct comparisons were made of these phenotypes carried out in eggs. Reassortants made from the Russian live attenuated influenza donor strains A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69 were prepared entirely in MDCK cells and their phenotypes evaluated using the MDCK cell-based assay. It is concluded that MDCK cells are more sensitive than eggs for the measurement of ts and ca phenotype of influenza viruses (particularly for influenza A) and they provide an alternative means for screening candidate reassortants prior to determining their genome composition. PMID:14715309

Kiseleva, I; Su, Q; Toner, T J; Szymkowiak, C; Kwan, W-S; Rudenko, L; Shaw, A R; Youil, R

2004-03-01

358

Identification of cancer stem-like side population cells in purified primary cultured human laryngeal squamous cell carcinoma epithelia.  

PubMed

Cancer stem-like side population (SP) cells have been identified in many solid tumors; however, most of these investigations are performed using established cancer cell lines. Cancer cells in tumor tissue containing fibroblasts and many other types of cells are much more complex than any cancer cell line. Although SP cells were identified in the laryngeal squamous cell carcinoma (LSCC) cell line Hep-2 in our pilot study, it is unknown whether the LSCC tissue contains SP cells. In this study, LSCC cells (LSCCs) were primary cultured and purified from a surgically resected LSCC specimen derived from a well-differentiated epiglottic neoplasm of a Chinese male. This was followed by the verification of epithelium-specific characteristics, such as ultrastructure and biomarkers. A distinct SP subpopulation (4.45±1.07%) was isolated by Hoechst 33342 efflux analysis from cultured LSCCs by using a flow cytometer. Cancer stem cell (CSC)-associated assays, including expression of self-renewal and CSC marker genes, proliferation, differentiation, spheroid formation, chemotherapy resistance, and tumorigenicity were then conducted between SP and non-SP (NSP) LSCCs. In vitro and in vivo assays revealed that SP cells manifested preferential expression of self-renewal and CSC marker genes, higher capacity for proliferation, differentiation, and spheroid formation; enhanced resistance to chemotherapy; and greater xenograft tumorigenicity in immunodeficient mice compared with NSP cells. These findings suggest that the primary cultured and purified LSCCs contain cancer stem-like SP cells, which may serve as a valuable model for CSC research in LSCC. PMID:23776540

Wu, Chun-Ping; Zhou, Liang; Xie, Ming; Du, Huai-Dong; Tian, Jie; Sun, Shan; Li, Jin-Yan

2013-01-01

359

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

360

Culture surface influence on T-cell phenotype and function.  

PubMed

When dealing with T lymphocyte culture there is currently very less information available about the interaction between T-cells and the culture system. In this study we look at the influence of the culture chamber on T-cell proliferation in two main aspects of the culture system, namely: culture chamber material and geometry. The study was carried out using unique polymeric closed cell culture inserts, which were processed via injection moulding from polystyrene (PS), polycarbonate (PC), polyetherurethane (PEU), polystyrene-co-acrylonitrile (PSAN) and polyetherimide (PEI). Furthermore culture chamber geometry was studied using commercially available 24, 12 and 6-well plates prepared from tissue culture plastic (TCP). For T lymphocyte stimulation two methods were used involving either EBV peptide pools or MACS iBead particles depending on the experiment performed. Culture was done with 1645 RPMI medium supplemented with foetal calf serum, penicillin, streptomycin and rhIL-2. We found four materials out of five we tested (PS, PC, PSAN and PEI) exhibited similar fold expansions with minimal influence on proportions of CD4 and CD8, while PEU had a negative influence on T cell growth along with adversely affected CD4/CD8 proportions. Changes in the geometry of TCP had no effect on T cell growth or maturation rather the size of geometry seems to have more influence on proliferation. T-cells appear to prefer smaller geometries during initial stages of culture while towards the end of the culture size becomes less significant to cell proliferation. The parameters tested in this study have significant influences on T-cell growth and are necessary to consider when designing and constructing expansion systems for antigen specific T lymphocytes. This is important when culturing T-cells for immunotherapeutic applications where antigen specificity, T-cell maturation and function should remain unaffected during culture. PMID:24099989

Hashimdeen, Shaikh Shimaz; Römhild, Andy; Schmueck, Michael; Kratz, Karl; Lendlein, Andreas; Kurtz, Andreas; Reinke, Petra

2013-01-01

361

Stem cell properties in cell cultures from different stage of melanoma progression.  

PubMed

Cutaneous melanoma is an extremely heterogenous human cancer. The most aggressive melanoma may contain deregulated cells with undifferentiated/stem cell-like phenotype. A critical mechanism by which melanoma cells enhance their invasive capacity is the dissolution of the intercellular adhesion and the acquisition of mesenchymal features as a part of an epithelial-to-mesenchymal transition. The aim of this study was to clarify the role of a stem cell-like population in human melanomas by means of melanocytic cell culture analysis obtained from distinct histotypes of primary and metastatic malignant melanoma. Patients with advanced melanoma >2 cm in diameter and/or >300 mm surface were enrolled. The melanoma cells were isolated from skin biopsies of lentigo maligna melanoma, superficial spreading melanoma, nodular melanoma, and metastatic melanoma. The colony forming unit assay and alkaline phosphatase stain were evaluated. Cells were subsequently cultured and maintained in different media to evaluate their ability to differentiate into osteogenic and adipogenic lineages. Immunohistochemistry and flow cytometry analysis were performed to evaluate antigenic markers CD90, CD73, CD105, CD146, CD20, CD166, and Nestin. This study confirms that melanoma can include heterogenous cell populations with the ability both to self-renew and to a give rise to differentiated progeny. Melanoma cells displayed intratumoral heterogeneity and dynamic antigen phenotypes. Histologically, transitions from normal skin to melanoma were associated with a gradual increase in the expression of CD146, CD20, CD133, Nestin, and CD73. These molecular profiles could be further analyzed and, in the future, used for the development of novel biomolecular targeted-therapy approaches. PMID:23702651

Magnoni, Cristina; Giudice, Stefania; Pellacani, Giovanni; Bertazzoni, Giorgia; Longo, Caterina; Veratti, Eugenia; Morini, Daria; Benassi, Luisa; Vaschieri, Cristina; Azzoni, Paola; De Pol, Anto; Seidenari, Stefania; Tomasi, Aldo; Pollio, Annamaria; Ponti, Giovanni

2014-03-01

362

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

363

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

364

Development of a luciferase based viral inhibition assay to evaluate vaccine induced CD8 T-cell responses.  

PubMed

Emergence of SIV and HIV specific CD8 T cells has been shown to correlate with control of in vivo replication. Poor correlation between IFN-? ELISPOT responses and in vivo control of the virus has triggered the development of more relevant assays to assess functional HIV-1 specific CD8 T-cell responses for the evaluation and prioritization of new HIV-1 vaccine candidates. We previously established a viral inhibition assay (VIA) that measures the ability of vaccine-induced CD8 T-cell responses to inhibit viral replication in autologous CD4 T cells. In this assay, viral replication is determined by measuring p24 in the culture supernatant. Here we describe the development of a novel VIA, referred to as IMC LucR VIA that exploits replication-competent HIV-1 infectious molecular clones (IMCs) in which the complete proviral genome is strain-specific and which express the Renilla luciferase (LucR) gene to determine viral growth and inhibition. The introduction of the luciferase readout does provide significant improvement of the read out time. In addition to switching to the LucR read out, changes made to the overall protocol resulted in the miniaturization of the assay from a 48 to a 96-well plate format, which preserved sample and allowed for the introduction of replicates. The overall assay time was reduced from 13 to 8 days. The assay has a high degree of specificity, and the previously observed non-specific background inhibition in cells from HIV-1 negative volunteers has been reduced dramatically. Importantly, we observed an increase in positive responses, indicating an improvement in sensitivity compared to the original VIA. Currently, only a limited number of "whole-genome" IMC-LucR viruses are available and our efforts will focus on expanding the panel to better evaluate anti-viral breadth. Overall, we believe the IMC LucR VIA provides a platform to assess functional CD8 T-cell responses in large-scale clinical trial testing, which will enhance the ability to select the most promising HIV-1 vaccine candidates capable of controlling HIV-1 replication in vivo. PMID:24291126

Naarding, Marloes A; Fernandez, Natalia; Kappes, John C; Hayes, Peter; Ahmed, Tina; Icyuz, Mert; Edmonds, Tara G; Bergin, Philip; Anzala, Omu; Hanke, Tomas; Clark, Lorna; Cox, Josephine H; Cormier, Emmanuel; Ochsenbauer, Christina; Gilmour, Jill

2014-07-01

365

Direct gene transfer into human cultured cells facilitated by laser micropuncture of the cell membrane  

SciTech Connect

The selective alteration of the cellular genome by laser microbeam irradiation has been extensively applied in cell biology. We report here the use of the third harmonic (355 nm) of an yttrium-aluminum garnet laser to facilitate the direct transfer of the neo gene into cultured human HT1080-6TG cells. The resultant transformants were selected in media containing an aminoglycoside antibiotic, G418. Integration of the neo gene into individual chromosomes and expression of the gene were demonstrated by Southern blot analyses, microcell-mediated chromosome transfer, and chromosome analyses. The stability of the integrated neo gene in the transformants was shown by a comparative growth assay in selective and nonselective media. Transformation and incorporation of the neo gene into the host genome occurred at a frequency of 8x10-4-3x10-3. This method appears to be 100-fold more efficient than the standard calcium phosphate-mediated method of DNA transfer.

Tao, W.; Wilkinson, J.; Stanbridge, E.J.; Berns, M.W.

1987-06-01

366

Culturing and applications of rotating wall vessel bioreactor derived 3D epithelial cell models.  

PubMed

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2-D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment. Here, we describe a culture system that overcomes many of the culture condition boundaries of 2-D cell cultures, by using the innovative rotating wall vessel (RWV) bioreactor technology. We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues (1-6). The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions. The bioreactors come in two different formats, a high-aspect rotating vessel (HARV) or a slow-turning lateral vessel (STLV), in which they differ by their aeration source. Epithelial cells are added to the bioreactor of choice in combination with porous, collagen-coated microcarrier beads (Figure 1A). The cells utilize the beads as a growth scaffold during the constant free fall in the bioreactor (Figure 1B). The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-like characteristics often not observed under standard 2-D culture conditions (Figure 1D). These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo protein localization, and additional epithelial cell-type specific properties. The progression from a monolayer of epithelial cells to a fully differentiated 3-D aggregate varies based on cell type(1, 7-13). Periodic sampling from the bioreactor allows for monitoring of epithelial aggregate formation, cellular differentiation markers and viability (Figure 1D). Once cellular differentiation and aggregate formation is established, the cells are harvested from the bioreactor, and similar assays performed on 2-D cells can be applied to the 3-D aggregates with a few considerations (Figure 1E-G). In this work, we describe detailed steps of how to culture 3-D epithelial cell aggregates in the RWV bioreactor system and a variety of potential assays and analyses that can be executed with the 3-D aggregates. These analyses include, but are not limited to, structural/morphological analysis (confocal, scanning and transmission electron microscopy), cytokine/chemokine secretion and cell signaling (cytometric bead array and Western blot analysis), gene expression analysis (real-time PCR), toxicological/drug analysis and host-pathogen interactions. The utilization of these assays set the foundation for more in-depth and expansive studies such as metabolomics, transcriptomics, proteomics and other array-based applications. Our goal is to present a non-conventional means of culturing human epithelial cells to produce organotypic 3-D models that recapitulate the human in vivo tissue, in a facile and robust system to be used by researchers with diverse scientific interests. PMID:22491366

Radtke, Andrea L; Herbst-Kralovetz, Melissa M

2012-01-01

367

Evaluation of new immunochromatographic assay kit for adenovirus detection in throat swab: comparison with culture and real-time PCR results.  

PubMed

A new immunochromatographic (IC) assay kit, BD Veritor System Adeno was evaluated to comparing with commercial available kit, BD Adeno Examan, cell culture, and real-time PCR using throat swab samples. Specimens were collected from 146 pediatric patients between July 2011 and January 2012. Mean age of patients was 4 years (8 months-15 years old). Patients were diagnosed with pharyngitis (n = 67), tonsillitis (n = 45), pharyngoconjunctival fever (n = 26), upper respiratory tract infection (n = 6), conjunctivitis (n = 1), or bronchitis (n = 1). Thirty-one of the patients (21.2%) had more than one disease. Among all samples, 61 (41.8%) were positive for adenovirus with BD Veritor System Adeno; 68 (46.6%) with BD Adeno Examan; 63 (43.2%) with real-time PCR; and 65 (44.5%) with cell culture. Serotype 3 (n = 41; 63.1%) was predominant among the 65 adenovirus isolates, followed by serotype 2 (n = 12; 18.5%), 1 (n = 6; 9.2%), 5 (n = 4; 6.2%), and 4 (n = 2; 3.1%). Relative sensitivity and specificity of BD Veritor System Adeno, BD Adeno Examan, and real-time PCR were 93.8% and 98.7%, 96.9% and 93.8%, and 96.9% and 100%, respectively. Positive predictive and negative predictive values for these methods were 98.4% and 95.1%, 92.6% and 97.4%, and 100% and 97.6%, respectively. The sensitivity and specificity of real-time PCR was greater than that of IC assay kits. However, IC assay kits also showed high sensitivity and specificity appropriate for clinical use. PMID:24594452

Morozumi, Miyuki; Shimizu, Hideaki; Matsushima, Yuki; Mitamura, Keiko; Tajima, Takeshi; Iwata, Satoshi; Ubukata, Kimiko

2014-05-01

368

Detection of Brain Tumor Cells in the Peripheral Blood by a Telomerase Promoter-Based Assay  

PubMed Central

Blood tests to detect circulating tumor cells (CTC) offer great potential to monitor disease status, gauge prognosis, and guide treatment decisions for patients with cancer. For patients with brain tumors, such as aggressive glioblastoma multiforme, CTC assays are needed that do not rely on expression of cancer cell surface biomarkers like epithelial cell adhesion molecules that brain tumors tend to lack. Here, we describe a strategy to detect CTC based on telomerase activity, which is elevated in nearly all tumor cells but not normal cells. This strategy uses an adenoviral detection system that is shown to successfully detect CTC in patients with brain tumors. Clinical data suggest that this assay might assist interpretation of treatment response in patients receiving radiotherapy, for example, to differentiate pseudoprogression from true tumor progression. These results support further development of this assay as a generalized method to detect CTC in patients with cancer. PMID:24525740

MacArthur, Kelly M.; Kao, Gary D.; Chandrasekaran, Sanjay; Alonso-Basanta, Michelle; Chapman, Christina; Lustig, Robert A.; Wileyto, E. Paul; Hahn, Stephen M.; Dorsey, Jay F.

2014-01-01

369

3D inverted colloidal crystals in realistic cell migration assays for drug screening applications.  

PubMed

Screening drugs for their specific impact on cell mechanics, in addition to targeting adhesion and proteolysis, will be important for successfully moderating migration in infiltrative disorders including cancer metastasis. We present 3D inverted colloidal crystals made of hydrogel as a realistic cell migration assay, where the geometry and stiffness can be set independently to mimic the tissue requirements in question. We show the utility of this 3D assay for drug screening purposes, specifically in contrast to conventional 2D migration studies, by surveying the effects of commonly used cytoskeletal toxins that impact cell mechanics. This assay allows studying large cell numbers for good statistics but at single-cell resolution. PMID:22038190

da Silva, Joakim; Lautenschläger, Franziska; Kuo, Cheng-Hwa R; Guck, Jochen; Sivaniah, Easan

2011-12-01

370

Cloning assay thresholds on cells exposed to ultrafast laser pulses  

NASA Astrophysics Data System (ADS)

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

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

1999-06-01

371

Cloning assay thresholds on cells exposed to ultrafast laser pulses  

NASA Astrophysics Data System (ADS)

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

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

1999-06-01

372

Preparation of Feeder plates for ES cell culture Gelatinize Tissue Culture Plates  

E-print Network

Preparation of Feeder plates for ES cell culture Gelatinize Tissue Culture Plates Gelatinize plates Cells with Mitomycin C 1. Aspirate off media from the STO cell plate. 2. Mix 120 µl of mitomycin C (0.5 mg/ml in PBS) with 6 ml of STO media. 3. Add to the STO cells (6 ml/10 cm) and incubate at 37°C

Oliver, Douglas L.

373

An Optically Controlled 3D Cell Culturing System  

PubMed Central

A novel 3D cell culture system was developed and tested. The cell culture device consists of a microfluidic chamber on an optically absorbing substrate. Cells are suspended in a thermoresponsive hydrogel solution, and optical patterns are utilized to heat the solution, producing localized hydrogel formation around cells of interest. The hydrogel traps only the desired cells in place while also serving as a biocompatible scaffold for supporting the cultivation of cells in 3D. This is demonstrated with the trapping of MDCK II and HeLa cells. The light intensity from the optically induced hydrogel formation does not significantly affect cell viability. PMID:22701475

Ishii, Kelly S.; Hu, Wenqi; Namekar, Swapnil A.; Ohta, Aaron T.

2012-01-01

374

Boron Deficiency in Cultured Pine Cells 1  

PubMed Central

A pronounced interaction between calcium, magnesium, and boron was found in growth studies with Pinus radiata cell cultures. Quantitative isoactivity data for the interaction was analyzed in terms of selected simple and plausible theoretical models. The data was found to be consistent with a model in which a critical acceptor molecule is activated only by binding both Ca and B at separate sites; Mg competitively displaces Ca to inactivate the acceptor. It was found that B is, surprisingly, not bound strongly (Kdiss = 450 ± 80 micromolar) and that the affinity for Ca is two orders of magnitude stronger than for Mg. Therefore only a small proportion of the acceptor will be boronated under natural conditions. Moderate levels of mannitol were found to aggravate B deficiency due to its effective removal by direct chemical complexation. At higher concentrations of mannitol (or other sugars), where osmotic contribution is significant, little B was needed to overcome growth inhibition—a result consistent with B having a primary role in cell wall biosynthesis. PMID:16667559

Teasdale, Robert Dixon; Richards, Dianne Katherine

1990-01-01

375

Fluorescent speckle microscopy in cultured cells.  

PubMed

After slightly more than a decade since it was first established, fluorescent speckle microscopy (FSM) has been intensively used to investigate macromolecular dynamics, such as microtubule flux in mitosis and meiosis, microtubule translocation in neurons, microtubule-binding proteins, and focal adhesion proteins, as well as the assembly of actin filaments. This state-of-the-art technique is based on nonuniform distribution of fluorescently labeled subunits diluted in the endogenous, unlabeled ones, resulting in microscopy-detectable speckled patterns. In order to enable sufficient contrast between neighboring diffraction-limited image regions, a low ratio between labeled and endogenous molecules is required, which can be achieved either by microinjection or by expression of limited amounts of fluorescently labeled subunits in cells. Over the years, the initial settings for FSM have been significantly improved by introduction of more sensitive cameras and spinning-disk confocal units, as well as by the development of specialized algorithms for image analysis. In this chapter, we describe our current FSM setup and detail on the necessary experimental approaches for its use in cultured cells, while discussing the present and future challenges of this powerful technique. PMID:22264533

Barisic, Marin; Pereira, António J; Maiato, Helder

2012-01-01

376

Apoptosis in batch cultures of Chinese Hamster Ovary cells  

Microsoft Academic Search

One of the main problems in the culture of Chinese Hamster Ovary (CHO) cells continues to be the inability to maintain the viability of the cultures over an extended period of time. The rapid decline in viability at the end of the culture is exacerbated by the absence of serum. In trying to reduce the extent of death in these

J. Goswami; A. J. Sinskey; H. Steller; G. N. Stephanopoulos; D. I. C. Wang

1999-01-01

377

A method for culturing human hair follicle cells.  

PubMed

For the first time a method for culturing human hair follicle cells is described. The bovine eye lens capsule, a basement membrane-like structure, is used as the substrate for the cultures. In a culture medium supplemented with hydrocortisone and insulin about 70% of the original follicles will form growing colonies of diploid keratinocytes. PMID:7006669

Weterings, P J; Vermorken, A J; Bloemendal, H

1981-01-01

378

Lipids trigger changes in the elasticity of the cytoskeleton in plant cells: a cell optical displacement assay for live cell measurements  

Microsoft Academic Search

An assay has been developed to quantita- tively measure the tension and elasticity of the cytoskeleton in living plant cells. The cell optical dis- placement assay (CODA) uses a focused laser beam to optically trap and displace transvacuolar and cortical strands through a defined distance within the cell. Results from these experiments provide evidence for the classification of at least

Sharon Grabski; Xiao Guang Xie; John E Holland; Melvin Schindler

1994-01-01

379

Development of a rapid cell-fusion-based phenotypic HIV-1 tropism assay  

PubMed Central

Introduction A dual split reporter protein system (DSP), recombining Renilla luciferase (RL) and green fluorescent protein (GFP) split into two different constructs (DSP1–7 and DSP8–11), was adapted to create a novel rapid phenotypic tropism assay (PTA) for HIV-1 infection (DSP-Pheno). Methods DSP1–7 was stably expressed in the glioma-derived NP-2 cell lines, which expressed CD4/CXCR4 (N4X4) or CD4/CCR5 (N4R5), respectively. An expression vector with DSP8–11 (pRE11) was constructed. The HIV-1 envelope genes were subcloned in pRE11 (pRE11-env) and transfected into 293FT cells. Transfected 293FT cells were incubated with the indicator cell lines independently. In developing the assay, we selected the DSP1–7-positive clones that showed the highest GFP activity after complementation with DSP8–11. These cell lines, designated N4R5-DSP1–7, N4X4-DSP1–7 were used for subsequent assays. Results The env gene from the reference strains (BaL for R5 virus, NL4-3 for X4 virus, SF2 for dual tropic virus) subcloned in pRE11 and tested, was concordant with the expected co-receptor usage. Assay results were available in two ways (RL or GFP). The assay sensitivity by RL activity was comparable with those of the published phenotypic assays using pseudovirus. The shortest turnaround time was 5 days after obtaining the patient's plasma. All clinical samples gave positive RL signals on R5 indicator cells in the fusion assay. Median RLU value of the low CD4 group was significantly higher on X4 indicator cells and suggested the presence of more dual or X4 tropic viruses in this group of patients. Comparison of representative samples with Geno2Pheno [co-receptor] assay was concordant. Conclusions A new cell-fusion-based, high-throughput PTA for HIV-1, which would be suitable for in-house studies, was developed. Equipped with two-way reporter system, RL and GFP, DSP-Pheno is a sensitive test with short turnaround time. Although maintenance of cell lines and laboratory equipment is necessary, it provides a safe assay system without infectious viruses. With further validation against other conventional analyses, DSP-Pheno may prove to be a useful laboratory tool. The assay may be useful especially for the research on non-B subtype HIV-1 whose co-receptor usage has not been studied much. PMID:24050252

Teeranaipong, Phairote; Hosoya, Noriaki; Kawana-Tachikawa, Ai; Fujii, Takeshi; Koibuchi, Tomohiko; Nakamura, Hitomi; Koga, Michiko; Kondo, Naoyuki; Gao, George F; Hoshino, Hiroo; Matsuda, Zene; Iwamoto, Aikichi

2013-01-01

380

Conversion of viable but nonculturable enteric bacteria to culturable by co-culture with eukaryotic cells.  

PubMed

Viable but nonculturable (VBNC) Vibrio cholerae non-O1/non-O139, V. parahaemolyticus, enterohemorrhagic Escherichia coli, enterotoxigenic E. coli, enteropathogenic E. coli, Shigella flexneri, and Salmonella enterica were converted to the culturable state by co-culture with selected eukaryotic cells, e.g., HT-29, Caco-2, T84, HeLa, Intestine 407, and CHO cells. PMID:22537150

Senoh, Mitsutoshi; Ghosh-Banerjee, Jayeeta; Ramamurthy, Thandavarayan; Colwell, Rita R; Miyoshi, Shin-Ichi; Nair, G Balakrish; Takeda, Yoshifumi

2012-05-01