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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

Development of an Easy and High-Throughput Cell Assay System with a Culture Chip and an Assay Chip  

NASA Astrophysics Data System (ADS)

High throughput cell assay is significantly important in drug screening, assessment of toxicity etc. Cell assay with a microchip is one of the candidates for high throughput cell assay. However, reported cell assay system with the microchip requires expensive apparatus for refluxing medium and investigation of optimum experimental condition for steady data. For an inexpensive, easy and high throughput cell assay, we introduce a new cell assay system combined with a culture chip and an assay chip made of poly(dimethyl siloxane). Cell culture chips enabled cell to proliferate along the microchannel without refluxing medium and permitted to prepare cell patterning easily. Also, assay chips formed concentration gradient inside the chip and allowed the cell assay with different concentrations of drug at the same time. Thus, our developed cell assay system can overcome the problems of the present cell assay and would promote the drug discovery, assessment of toxicity etc.

Sugiura, Kanako; Kaji, Noritada; Okamoto, Yukihiro; Tokeshi, Manabu; Baba, Yoshinobu

3

Defining cell culture conditions to improve human norovirus infectivity assays.  

PubMed

Significant difficulties remain for determining whether human noroviruses (hNoV) recovered from water, food, and environmental samples are infectious. Three-dimensional (3-D) tissue culture of human intestinal cells has shown promise in developing an infectivity assay, but reproducibility, even within a single laboratory, remains problematic. From the literature and our observations, we hypothesized that the common factors that lead to more reproducible hNoV infectivity in vitro requires that the cell line be (1) of human gastrointestinal origin, (2) expresses apical microvilli, and (3) be a positive secretor cell line. The C2BBe1 cell line, which is a brush-border producing clone of Caco-2, meets these three criteria. When challenged with Genogroup II viruses, we observed a 2 Log(10) increase in viral RNA titer. A passage experiment with GII viruses showed evidence of the ability to propagate hNoV by both quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microscopy. In our hands, using 3-D C2BBe1 cells improves reproducibility of the infectivity assay for hNoV, but the assay can still be variable. Two sources of variability include the cells themselves (mixed phenotypes of small and large intestine) and initial titer measurements using qRT-PCR that measures all RNA vs. plaque assays that measure infectious virus. PMID:23306266

Straub, T M; Hutchison, J R; Bartholomew, R A; Valdez, C O; Valentine, N B; Dohnalkova, A; Ozanich, R M; Bruckner-Lea, C J

2013-01-01

4

[Cytomegalovirus isolation by conventional cell culture and shell vial assay].  

PubMed

In immunocompromised patients, diagnosis of Cytomegalovirus (CMV) active infection is of utmost importance for the initiation, monitoring and ending of antiviral therapy. Therefore, the presence of viral replication should be demonstrated. Isolation in tissue culture is one of the standard methods. The objective of the present paper was to compare two isolation procedures for CMV: conventional cell culture (CC) and rapid shell vial (SV) assay in human fibroblasts. A total of 584 clinical samples were studied between 1991 and 1998. CMV was isolated in 14.4% of the samples, 11.8% of which were positive by SV and 7.7% by CC. Out of 84 positive samples, concordance between both methods was observed in 36% of the cases. We found that 46% of the samples were positive only by SV, while 18% were positive only by CC. The average time required for obtaining the results by CC was 22.6 +/- 2.3 days. Out of the 69 samples positive by SV, 43% were already positive after 24 hours and the rest after 48 hours. These results indicate that SV was more sensitive and rapid than CC. The main advantage of CC, despite its time-consuming process, is the ability to recover the viral strain for both antiviral susceptibility phenotypical tests and strain characterization. Furthermore, in this study, absence of CC would have resulted in the loss of 18% of the positive diagnoses. In conclusion, simultaneous use of both methods is suggested in order to obtain a rapid result and the highest sensitivity. PMID:11808422

Galiano, M C; Videla, C M; Sánchez Puch, S; Carballal, G

2001-01-01

5

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

Stephens, D S

1989-01-01

6

Assaying prions in cell culture: the standard scrapie cell assay (SSCA) and the scrapie cell assay in end point format (SCEPA).  

PubMed

Prions are usually quantified by bioassays based on intracerebral inoculation of animals, which are slow, imprecise, and costly. We have developed a cell-based prion assay that is based on the isolation of cell lines highly susceptible to certain strains (Rocky Mountain Laboratory and 22L) of mouse prions and a method for identifying individual, prion-infected cells and quantifying them. In the standard scrapie cell assay (SSCA), susceptible cells are exposed to prion-containing samples for 4 days, grown to confluence, passaged two or three times, and the proportion of rPrP(Sc)-containing cells is determined with automated counting equipment. The dose response is dynamic over 2 logs of prion concentrations. The SSCA has a standard error of +/-20-30%, is as sensitive as the mouse bioassay, 10 times faster, at least 2 orders of magnitude less expensive, and it is suitable for robotization. Assays performed in a more time-consuming end point titration format extend the sensitivity and show that infectivity titers measured in tissue culture and in the mouse are similar. PMID:18576147

Mahal, Sukhvir P; Demczyk, Cheryl A; Smith, Emery W; Klohn, Peter-Christian; Weissmann, Charles

2008-01-01

7

Evaluation of a rat tracheal epithelial cell culture assay system to identify respiratory carcinogens  

Microsoft Academic Search

To evaluate a short-term epithelial cell assay system to detect respiratory carcinogens, primary cultures of rat tracheal epithelial cells were exposed to a series of 17 compounds and scored for morphologically transformed cell colonies 28 days later. The test compounds included known carcinogens and noncarcinogens in volatile or liquid form. Tracheal epithelial cells were isolate from F344 rats, plated onto

Vernon E. Steele; Julia T. Arnold; John Van Arnold; Marc J. Mass

1989-01-01

8

A colorimetric assay for determination of cell viability in algal cultures  

Microsoft Academic Search

In this work, we propose the determination of cell viability in algal cultures by using a colorimetric assay widely used for estimation of cell proliferation in animal cell cultures. The method is based on in vivo reduction by metabolically active cells of a tetrazolium compound (MTS=3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium, inner salt) to a colored formazan, with maximal absorbance at 490 nm, that is

Juan M Capasso; Belén R Coss??o; Tomás Berl; Christopher J Rivard; Carlos Jiménez

2003-01-01

9

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

10

A colorimetric assay for determination of cell viability in algal cultures.  

PubMed

In this work, we propose the determination of cell viability in algal cultures by using a colorimetric assay widely used for estimation of cell proliferation in animal cell cultures. The method is based on in vivo reduction by metabolically active cells of a tetrazolium compound (MTS=3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium, inner salt) to a colored formazan, with maximal absorbance at 490 nm, that is released to the culture medium. For this purpose, we have tested two microalgae with high commercial value (Dunaliella and Spirulina) and two seaweeds with different morphology (Ulva and Gracilaria). Color development in this assay is directly proportional to the number of viable cells, to the incubation time in the presence of the assay solution, and to the incubation temperature. A direct significant correlation was found between algal photosynthesis rate and color development in all species used through this work. Moreover, the intensity of absorbance at 490 nm was significantly lower in stressed cells (e.g. in nutrient-limited cultures, in the presence of toxic substances, and in osmotically-stressed cultures). We conclude that cell viability of algal cultures can be rapidly and easily estimated through colorimetric determination of the reduction of MTS to formazan. PMID:12919790

Capasso, Juan M; Cossío, Belén R; Berl, Tomás; Rivard, Christopher J; Jiménez, Carlos

2003-07-01

11

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

PubMed

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

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

2014-01-01

12

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

EPA Science Inventory

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

13

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.

Nickson, Catherine M.; Parsons, Jason L.

2014-01-01

14

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

15

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.

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

2014-01-01

16

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

PubMed Central

Cryptosporidium parvum represents a challenge to the water industry and a threat to public health. In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of C. parvum with disinfectants. The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine. The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst. Effective oocyst inactivation was achieved (>2 log10 units) with LP-UV (20 mJ/cm2) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with <0.1 log10 unit being inactivated. These results demonstrate the inability of MIOX to inactivate Cryptosporidium. The assay is a valuable tool for the evaluation of disinfection systems for drinking water and recycled water.

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

2003-01-01

17

Single-cell assays  

PubMed Central

This review presents an overview of literature that describes the applications of microfluidics to assay individual cells. We quantify the content of an individual mammalian cell, so that we can understand what criteria a single-cell assay must satisfy to be successful. We put in context the justification for single-cell assays and identify the characteristics that are relevant to single-cell assays. We review the literature from the past 24 months that describe the methods that use microfabrication—conventional or otherwise—and microfluidics in particular to study individual cells, and we present our views on how an increasing emphasis on three-dimensional cell culture and the demonstration of the first chemically defined cell might impact single-cell assays.

Ryan, Declan; Ren, Kangning; Wu, Hongkai

2011-01-01

18

Optimization of the BGM cell line culture and viral assay procedures for monitoring viruses in the environment.  

PubMed Central

An in-depth study of the continuous cell line designated BGM is described herein, and recommendations are made for standardizing cell culture and viral assay procedures. Based on data gathered from a survey of 58 laboratories using this cell line, a research plan was developed that included the study of growth media, sera, NaHCO3 levels, culture bottles, cell concentration, overlay media, agar, virus infection conditions, and cell-dissociating agents. Additionally, a comparative virus isolation study with BGM cells and nine other cell types was conducted with 37 sewage samples collected from nine different geographic areas. The results of the study indicated that the BGM cell line is superior for virus isolation when compared with the other cell types and that certain media and additives tend to increase BGM cell sensitivity to a specific group of viruses. A standardized procedure for cultivation of BGM cells is described which provides a more effective enterovirus assay system.

Dahling, D R; Wright, B A

1986-01-01

19

RT-PCR and cell culture infectivity assay to detect enteroviruses during drinking water treatment processes.  

PubMed

In this study, 62 water samples were collected from two water treatment plants (WTPs) in Suez Canal cities (Port Said and Ismaillia) and one plant in Cairo (Giza WTP) in addition to the beginning of the two Nile river branches (Rosetta and Damietta). Viruses were concentrated by adsorption-elution ethod sing 142 mm-diameter nitrocellulose membrane of 0.45 microm pore size and eluted with 3% beef extract at pH 9.5. The concentrated samples were inoculated for 3 successive passages in three cell culture types (Vero, BGM and RD). Enterovirus RNAs in CPE-induced samples were extracted by guanidinium thiocyanate/ phenol/chloroform and heat shock methods and detected by RT-PCR and neutralization test. The results showed that eight samples [14.5% (8/62)] contained enteroviruses most of them were polioviruses [87.5% (7/8)] and coxsackievirus type B2 [12.5% (1/8)]. The three cell cultures were of the same sensitivity to detect the isolated viruses. Also, RT-PCR followed by neutralization assay facilitates and accelerate the results. The guanidinium thiocyanate extraction method was more sensitive than heat shock method. The results turned our attention to review our technology of water treatment and disinfection step in addition to the selection of suitable intake for the drinking water treatment plants. PMID:17219867

Ali, M A; El-Esnawy, N A; Shoaeb, A R; Ibraheim, M; El-Hawaary, S E

1999-01-01

20

Accurate non-invasive image-based cytotoxicity assays for cultured cells  

Microsoft Academic Search

BACKGROUND: The CloneSelect™ Imager system is an image-based visualisation system for cell growth assessment. Traditionally cell proliferation is measured with the colorimetric MTT assay. RESULTS: Here we show that both the CloneSelect Imager and the MTT approach result in comparable EC50 values when assaying the cytotoxicity of cisplatin and oxaliplatin on various cell lines. However, the image-based technique was found

Patricia Marqués-Gallego; Hans den Dulk; Claude Backendorf; Jaap Brouwer; Jan Reedijk; Julian F Burke

2010-01-01

21

Plasminogen activator in cultured Lewis lung carcinoma cells measured by chromogenic substrate assay  

Microsoft Academic Search

A chromogenic substrate assay for the plasminogen activator (PA) activity of Lewis lung carcinoma cells has been developed. The cells were incubated with plasminogen, the activation of which to plasmin was measured by the amidolysis of the chromogenic substrate S-2251. This was routinely performed as a 4h serum-free assay, but a variation lasting 24 h, in medium supplemented with plasminogen-free

P Whur; M Magudia; J Boston; J Lockwood; D C Williams

1980-01-01

22

Rapid prenatal and postnatal detection of inborn errors of propionate, methylmalonate, and cobalamin metabolism: A sensitive assay using cultured cells  

Microsoft Academic Search

A sensitive, reliable, and easily performed procedure is described for the prenatal and postnatal detection of inborn errors of propionate, methylmalonate, and cobalamin metabolism using cultured amniotic cells and skin fibroblasts. With this assay, control fibroblast lines incorporated a mean of 6.89 nanoatoms 14C\\/mg protein from [1-14C]propionate into trichloroacetic acid (TCA)-precipitable cell material in 10h. Twenty-five mutant fibroblast lines from

Huntington F. Willard; Lalit M. Ambani; Anita C. Hart; Maurice J. Mahoney; Leon E. Rosenberg

1976-01-01

23

Sensitive assay of RNA interference in Drosophila and Chinese hamster cultured cells using firefly luciferase gene as target  

Microsoft Academic Search

A sensitive cellular assay system for RNA interference was developed using the firefly luciferase gene as target. RNA interference was noted not only in Drosophila cultured cells but Chinese hamster cells (CHO-K1) as well, although double-stranded RNA required for the latter was 2500 times more than for the former. Cognate double-stranded RNA as short as 38 bp was found to

Kumiko Ui-Tei; Shuhei Zenno; Yuhei Miyata; Kaoru Saigo

2000-01-01

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

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

1977-01-01

25

Human cultured dendritic cells show differential sensitivity to chemotherapy agents as assessed by the MTS assay  

Microsoft Academic Search

Assessment of the chemosensitivity of dendritic cells (DC) may allow more rational development of combined chemotherapy and immunotherapy protocols. Human monocyte-derived DC generated reproducible results in the MTS (Owen’s reagent) assay, which was then used to study DC survival after treatment with four different chemotherapy agents. DC preparations from three different donors were used per drug. DC were sensitive to

D Chao; P Bahl; S Houlbrook; L Hoy; A L Harris; J M Austyn

1999-01-01

26

A simple and rapid reverse transcriptase assay for the detection of retroviruses in cell cultures  

Microsoft Academic Search

Reverse transcriptase (RT) is a good diagnostic tool for the detection of retroviruses. We have developed a simple and rapid\\u000a assay for RT activity in culture supernatants. A 370-base RNA sequence from the tetracycline-resistance gene in pBR322 plasmid\\u000a DNA was used as a template for RT-mediated cDNA synthesis. To detect the resultant cDNA, we used the nested polymerase chain\\u000a reaction.

Haruhiko Kuno; Hiromi Ikeda; Masao Takeuchi; Touho Yoshida

1999-01-01

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

Polymer-based mesh as supports for multi-layered 3D cell culture and assays.  

PubMed

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

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

2014-01-01

29

An integrated cell culture and reverse transcription quantitative PCR assay for detection of infectious rotaviruses in environmental waters.  

PubMed

Rotaviruses exist widely in water environments and are the major cause to the gastroenteritis in children. To overcome the limitations associated with the current methods for detecting rotaviruses in environmental samples, such as long duration with the traditional cell culture-based plaque assay, inability to detect infectivity with RT-PCR-based molecular methods and lower sensitivity with ELISA tests, we developed an integrated cell culture and reverse transcription quantitative PCR (ICC-RT-qPCR) assay to detect infectious rotaviruses based on detection of viral RNA during replication in cells. The cell culturing step before qPCR allows the infectious rotaviruses to replicate and be detected because they are the only ones that can infect cells and produce RNA. The results showed that as low as 0.2 PFU/ml rotaviruses were detected by ICC-RT-qPCR after 2 days of incubation. With samples, the copy numbers of VP7 gene of rotaviruses linearly correlated (with a coefficient (R(2)) of 0.9575) with initial virus concentrations ranging from 0.2 to 200 PFU/ml. In parallel comparing tests, the ICC-RT-qPCR exhibited higher sensitivity than both the plaque assay and the RT-qPCR when applied to field samples. ICC-RT-qPCR detected infectious rotavirus in 42% (10/24) of secondary effluents, while only 21% (5/24) and 12% (3/24) of samples were positive with either the plaque counting or the RT-qPCR method, respectively. Concentrations of rotaviruses in secondary effluent samples were determined to be 1-30 PFU/l. The results demonstrated that the developed ICC-RT-qPCR method reduced test duration and improved sensitivity towards infectious rotavirus and therefore can be an effective and quantitative tool for detecting infectious rotaviruses in water environments. PMID:20399813

Li, Dan; Gu, April Z; Yang, Wan; He, Miao; Hu, Xiu-Hua; Shi, Han-Chang

2010-07-01

30

Tissue culture assays using Caco-2 cell line differentiate virulent from non-virulent Listeria monocytogenes strains.  

PubMed

Within the group of Listeria sp., only L. monocytogenes is pathogenic for humans and numerous studies of L. monocytogenes strains have described non-virulent isolates. In this study, the potential value of two tissue culture assays (TCA) was analysed to ascertain the virulence properties of L. monocytogenes strains, initially typed for virulence using the immunocompromised mouse model (ICMM). The first assay assessed both the penetration into, and multiplication within, Caco-2 cells (PM assay): the second was a plaque-forming assay (PF assay). All the clinical isolates (nine strains) were virulent in both TCA. Conversely, all the non-pathogenic species (seven strains) were non-virulent in PM and PF assays. Compared with the virulence obtained in the ICMM with 29 Listeria strains, including 12 non-virulent L. monocytogenes strains, the sensitivity of both TCA was equal to1. Specificity was 0.89 and 0.84 for the PF and PM assays, respectively. However, a study of strains exhibiting virulence differences in three other in vivo virulence models showed that ICMM only detected highly virulent strains. The specificity of the PF test could, therefore, be higher, and close to that obtained by the enumeration of viable bacteria in the spleen of mice infected by subcutaneous injection in the footpad and by intravenous injection. Taken together, this study confirms the existence of low-virulence L. monocytogenes strains and shows that the virulence status of some non-clinical L. monocytogenes isolates depends on the virulence models used. The data suggest that the PF assay could be used as a primary test to evaluate the virulence of Listeria strains in order to reduce the cost of testing all strains in vivo. PMID:9750307

Van Langendonck, N; Bottreau, E; Bailly, S; Tabouret, M; Marly, J; Pardon, P; Velge, P

1998-08-01

31

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?  

PubMed Central

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) and soft (n = 131) stool specimens from 377 symptomatic patients, all testing was performed on the same day by independent laboratory staff according to the manufacturers' protocols. Toxigenic bacterial culture was performed as follows. A 0.5-ml aliquot of stool was heated to 80°C for 10 min, followed by inoculation onto modified cycloserine cefoxitin fructose agar with and without horse blood (Remel, Lenexa, KS) and into prereduced chopped-meat broth. Of the 404 stool specimens tested, 340 were negative and 40 were positive (10.0% prevalence) both by PCR for tcdB and by cytotoxin production. The overall agreement between the BD GeneOhm Cdiff assay and the TOX-B test was 94.8% (380/401). When the TOX-B test was used as the reference method, the initial sensitivity, specificity, and positive and negative predictive values of the BD GeneOhm Cdiff assay were 90.9% (40/44), 95.2% (340/357), 70.2% (40/57), and 98.8% (340/344), respectively. When toxigenic culture was used as the “gold standard,” the sensitivity, specificity, and positive and negative predictive values of the BD GeneOhm Cdiff assay were 83.6%, 98.2%, 89.5%, and 97.1%, respectively, and those of the TOX-B test were 67.2%, 99.1%, 93.2%, and 94.4%, respectively. PCRs for three samples were inhibited upon initial testing; one sample was resolved upon retesting. One sample produced nonspecific cytotoxin results. The BD GeneOhm Cdiff assay performed well compared to a standard cell culture neutralization assay and to toxigenic culture for the detection of toxigenic C. difficile directly from fecal specimens.

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

2009-01-01

32

Human cultured dendritic cells show differential sensitivity to chemotherapy agents as assessed by the MTS assay.  

PubMed

Assessment of the chemosensitivity of dendritic cells (DC) may allow more rational development of combined chemotherapy and immunotherapy protocols. Human monocyte-derived DC generated reproducible results in the MTS (Owen's reagent) assay, which was then used to study DC survival after treatment with four different chemotherapy agents. DC preparations from three different donors were used per drug. DC were sensitive to doxorubicin (concentration range 0.1-50 microM) with variation in sensitivity between donors (IC50 244-1100 nM). The most extreme variation was seen for vinblastine (concentration range 250-0.025 microM with IC50 0.15-17.25 microM). In contrast, there was relative resistance to etoposide (concentration range 0.2-200 microM) and 5-fluorouracil (concentration range 0.7-7700 microM) with no toxicity seen until 50 microM and 770 microM respectively. The function of DC in allogeneic mixed leucocyte reactions closely paralleled results from the MTS assays. The differential sensitivity to chemotherapy agents did not appear to be due to expression of P-glycoprotein. These results suggest that etoposide or 5-fluorouracil is less likely to reduce the immunotherapeutic potential of DC and may be valuable in the design of prodrug activation therapy. PMID:10604723

Chao, D; Bahl, P; Houlbrook, S; Hoy, L; Harris, A; Austyn, J M

1999-12-01

33

Comparison of BGM and PLC/PRC/5 Cell Lines for Total Culturable Viral Assay of Treated Sewage?  

PubMed Central

The objective of this study was to compare PLC/PRF/5 and BGM cell lines for use in a total culturable viral assay (TCVA) of treated sewage effluents. Samples were collected before and after chlorination from an activated sludge wastewater treatment plant and from the effluent of a high-rate enhanced flocculation system, followed by UV light disinfection. Cell monolayers were observed for cytopathic effect (CPE) after two passages of 14 days each. Monolayers exhibiting viral CPE were tested for the presence of adenoviruses and enteroviruses by PCR or reverse transcription-PCR. Eight percent of the samples exhibited CPE on BGM cells, and 57% showed CPE on PLC/PRF/5 cells. Only enteroviruses were detected on the BGM cells, while 30% and 52% of the samples were positive for enteroviruses and adenoviruses, respectively, on the PLC/PRF/5 cells. Thirty percent of the samples were positive for both adenoviruses and enteroviruses in chlorinated activated sludge effluent. Thirty percent of the samples were positive for adenoviruses in the UV treatment effluent, but no enteroviruses were detected. In conclusion, the PLC/PRF/5 cells were more susceptible than BGM cells to viruses found in treated sewage. The use of BGM cells for TCVA may underestimate viral concentration in sewage effluent samples. The PLC/PRF/5 cells were more susceptible to adenoviruses, which is important in the evaluation of UV disinfection systems because adenoviruses are highly resistant to UV inactivation.

Rodriguez, Roberto A.; Gundy, Patricia M.; Gerba, Charles P.

2008-01-01

34

Comparative sensitivity of the VecTest antigen-capture assay, reverse transcriptase-PCR, and cell culture for detection of West Nile virus in dead birds.  

PubMed

The sensitivity of the VecTest antigen-capture and RT-PCR assays were compared to brain tissue culture in Vero cells for the detection of WNV in a sample of dead birds collected in East Texas and southern Louisiana during the summer of 2003. Cell culture was used as the gold standard, because it yielded the most positives. Direct culture of brain tissue and the WNV antigen-capture assay, done on oropharyngeal swabs, gave statistically comparable results (46% and 40% positive, respectively). In contrast, RT-PCR done on brain homogenates was significantly less sensitive than direct tissue culture (33% compared to 46%, respectively). Results indicated that RT-PCR may not be consistently the most sensitive assay for detection of WNV in dead bird brain tissue and that the VecTest antigen-capture assay has a number of advantages over the other two detection techniques for routine surveillance activities. PMID:15631064

Siirin, Marina; Sargent, Chris; Langer, Rebecca C; Parsons, Ray; Vanlandingham, Dana L; Higgs, Stephen; Tesh, Robert B

2004-01-01

35

Geometric approach to segmentation and protein localization in cell culture assays.  

PubMed

Cell-based fluorescence imaging assays are heterogeneous and require the collection of a large number of images for detailed quantitative analysis. Complexities arise as a result of variation in spatial nonuniformity, shape, overlapping compartments and scale (size). A new technique and methodology has been developed and tested for delineating subcellular morphology and partitioning overlapping compartments at multiple scales. This system is packaged as an integrated software platform for quantifying images that are obtained through fluorescence microscopy. Proposed methods are model based, leveraging geometric shape properties of subcellular compartments and corresponding protein localization. From the morphological perspective, convexity constraint is imposed to delineate and partition nuclear compartments. From the protein localization perspective, radial symmetry is imposed to localize punctate protein events at submicron resolution. Convexity constraint is imposed against boundary information, which are extracted through a combination of zero-crossing and gradient operator. If the convexity constraint fails for the boundary then positive curvature maxima are localized along the contour and the entire blob is partitioned into disjointed convex objects representing individual nuclear compartment, by enforcing geometric constraints. Nuclear compartments provide the context for protein localization, which may be diffuse or punctate. Punctate signal are localized through iterative voting and radial symmetries for improved reliability and robustness. The technique has been tested against 196 images that were generated to study centrosome abnormalities. Corresponding computed representations are compared against manual counts for validation. PMID:17286692

Raman, S; Maxwell, C A; Barcellos-Hoff, M H; Parvin, B

2007-01-01

36

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.

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

2012-01-01

37

Cytotoxicity of mycotoxins evaluated by the MTT-cell culture assay  

Microsoft Academic Search

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

Monika Hanelt; Manfred Gareis; Birgit Kollarczik

1994-01-01

38

Quantification of neuron survival in monolayer cultures using an enzyme-linked immunosorbent assay approach, rather than by cell counting  

Microsoft Academic Search

The determination of neurotoxicity in monolayer mixed cultures has traditionally necessitated the time consuming and subjective procedure of counting neurons. In this paper, we propose a modification of an immunohistochemical staining method with a neuron-specific antibody against MAP2, that allows for quantification of neuron number to be done using an enzyme-linked immunosorbent assay (ELISA) plate reader. This new procedure involves

Sheila M. Brooke; Tonya M. Bliss; Laura R. Franklin; Robert M. Sapolsky

1999-01-01

39

Use of the MTT assay for estimating toxicity in primary astrocyte and C6 glioma cell cultures  

Microsoft Academic Search

Primary cultures of rat cortical astrocytes were exposed to 25 neurotoxic and non-neurotoxic compounds for 24 hr over a concentration range of 0.001 to 1000 ?g\\/ml and the effects were quantified using the MTT assay. EC50 values were obtained for nine of the compounds in the tested range, while increases in MTT conversion were seen at sub-cytotoxic concentrations for 12

M. R. Cookson; C. Mead; S. M. Austwick; V. W. Pentreath

1995-01-01

40

Cell motility assays.  

PubMed

This report summarises practical aspects to measuring cell motility in culture. The methods described here were discussed at a 1-day European Tissue Culture Society (ETCS-UK) workshop organised by John Masters and Gareth E Jones that was held at University College London on 19th April 2007. PMID:18071914

Hague, Angela; Jones, Gareth E

2008-10-01

41

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

PubMed

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

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

2013-06-01

42

Comparison of RT-PCR assay and virus isolation in cell culture for the detection of alkhumra hemorrhagic fever virus.  

PubMed

Alkhumra hemorrhagic fever virus (AHFV) is an emerging flavivirus that was isolated originally from Saudi Arabia in 1994-1995. The main tests used for the detection of AHFV are the real time (rt) RT-PCR and virus isolation in cell culture. In the present study the detection of AHFV by rtRT-PCR was compared with virus isolation in BHK-21, HEp-2, and LLC-MK2 cell lines. AHFV suspensions grown in BHK-21, HEp-2, and LLC-MK2 cell lines were serially diluted 10-fold from 10(-1) to 10(-11) . Samples from each dilution were used to inoculate four cell culture tubes and were also examined by the rtRT-PCR for AHFV RNA. Fifteen non-inoculated cell culture samples (five from each cell line) were included blindly in both tests. Thus, a total of 132 AHFV-positive and 15 negative control samples were tested. The rtRT-PCR could detect the viral RNA in all diluted specimens up to and including the 10(-10) dilution (40 specimens for each cell line), whereas, cell cultures were positive in 70% of specimens for BHK-21, 65% for LLC-MK2, and 45% for HEp-2 at this dilution. None of the three cell cultures nor the rtRT-PCR was positive at 10(-11) dilution. The specificity and positive predictive values of virus isolation compared to rtRT-PCR were each 100%, whereas the negative predictive values were 29.4% for BHK-21, 26.3% for LLC-MK2, and 18.5% for HEp-2. In conclusion, the rtRT-PCR is more sensitive than virus isolation for detecting AHFV. J. Med. Virol. 86:1176-1180, 2014. © 2013 Wiley Periodicals, Inc. PMID:24249525

Madani, Tariq A; Abuelzein, El-Tayb M E; Azhar, Esam I; Al-Bar, Hussein M S; Abu-Araki, Huda; Ksiazek, Thomas G

2014-07-01

43

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

Microsoft Academic Search

The spreading of diseases through foods is a worldwide concern. Here, molecular and in vitro cell-culture assays were employed to characterize 63 Brazilian Listeria monocytogenes isolates (food, 47; clinical, 16). Serotype 4b was the most predominant (49%) followed by ½b (30%), ½a (10%), ½c (6%), 3c (3%) and 3b (2%). Ribotyping yielded 17 ribopatterns, which were grouped into four phylogenetic

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

2010-01-01

44

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

45

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

46

Development and validation of an integrated cell culture-qRTPCR assay for simultaneous quantification of coxsackieviruses, echoviruses, and polioviruses in disinfection studies.  

PubMed

This study demonstrated the applicability of integrated cell culture-quantitative RTPCR (ICC-qRTPCR) for the simultaneous quantification of coxsackievirus, echovirus, and poliovirus in disinfection studies. Buffalo green monkey cells were inoculated with a 10-fold dilution series of mixed enteroviruses and incubated prior to qRTPCR quantification. Optimal assay conditions included three post infection washes and a 24-hour post infection incubation period based on successful differentiation between infectious and noninfectious viruses and significant and consistent viral replication rates. Ultraviolet disinfection studies were performed to validate the ICC-qRTPCR assay. Using the optimized assay, three-log microbial inactivation was achieved at UV doses of 30-44, 28-42, and 28-29 mJ/cm(2) for coxsackievirus B6, echovirus 12, and poliovirus 1, respectively. These results compare favorably to side-by-side assessments using conventional cultural techniques and values previously reported in the literature. This indicates that ICC-qRTPCR is a practical alternative for the simultaneous quantification of enteroviruses in disinfection studies. PMID:20107264

Mayer, B K; Ryu, H; Gerrity, D; Abbaszadegan, M

2010-01-01

47

A rapid and simple MTT-based spectrophotometric assay for determining drug sensitivity in monolayer cultures  

Microsoft Academic Search

Summary A rapid and sensitive spectrophotometric assay for determining viability in monolayer cultured cell lines, with specific applications in normal and drug resistant cell line determinations, is described. The assay involves conversion of the tetrazolium salt MTT by viable proliferating cells to an insoluble product, purple formazan. The chief advantage of this assay is that it requires fewer cells than

Jeffrey M. Edmondson; Linda S. Armstrong; Andrew O. Martinez

1988-01-01

48

Ligand Accumulation in Autocrine Cell Cultures  

Microsoft Academic Search

Cell-culture assays are routinely used to analyze autocrine signaling systems, but quantitative experiments are rarely possible. To enable the quantitative design and analysis of experiments with autocrine cells, we develop a biophysical theory of ligand accumulation in cell-culture assays. Our theory predicts the ligand concentration as a function of time and measurable parameters of autocrine cells and cell-culture experiments. The

Michael I. Monine; Alexander M. Berezhkovskii; Elizabeth J. Joslin; H. Steven Wiley; Douglas A. Lauffenburger; Stanislav Y. Shvartsman

2005-01-01

49

Cell Culture Models for Neurotoxicology  

Microsoft Academic Search

A range of in vitro cell culture methods are available for neurotoxicology which typically represent one of the two predominant cell types present in the brain, neurons and glial cells. These systems can be used in a two tiered approach, whereby simple cytotoxic models reveal the gross effects of a drug or compound and, subsequently, more complex and subtle assays

Glyn Stacey; Barbara Viviani

2001-01-01

50

Simultaneous evaluation of cell viability by neutral red, MTT and crystal violet staining assays of the same cells  

Microsoft Academic Search

By combining three separate toxic assays, the neutral red (NR), MTT and crystal violet staining assays (CVS), we developed a convenient assay method, the NMC assay, in which the NR, MTT and CVS assays are performed consecutively on the same cultured HeLa cells. The NMC assay is performed as follows: the cultured HeLa cells are first treated with NR, and

K Chiba; K Kawakami; K Tohyama

1998-01-01

51

An optimized microplate assay system for quantitative evaluation of plant cell wall-degrading enzyme activity of fungal culture extracts  

Microsoft Academic Search

Developing enzyme cocktails for cellulosic bio- mass hydrolysis complementary to current cellulase systems is a critical step needed for economically viable biofuels production. Recent genomic analysis indicates that some plant pathogenic fungi are likely a largely untapped resource in which to prospect for novel hydrolytic enzymes for biomass conversion. In order to develop high throughput screening assays for enzyme bioprospecting,

Brian C. King; Marie K. Donnelly; Gary C. Bergstrom; Larry P. Walker; Donna M. Gibson

2009-01-01

52

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

PubMed

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

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

2012-04-01

53

A universal nanoparticle cell secretion capture assay.  

PubMed

Secreted proteins play an important role in intercellular interactions, especially between cells of the immune system. Currently, there is no universal assay that allows a simple noninvasive identification and isolation of cells based on their secretion of various products. We have developed such a method. Our method is based on the targeting, to the cell surface, of heterofunctional nanoparticles coupled to a cell surface-specific antibody and to a secreted protein-specific antibody, which captures the secreted protein on the surface of the producing cell. Importantly, this method does not compromise cellviability and is compatible with further culture and expansion of the secreting cells. PMID:22996967

Fitzgerald, Wendy; Grivel, Jean-Charles

2013-02-01

54

Correlation between Clostridium difficile Bacterial Load, Commercial Real-Time PCR Cycle Thresholds, and Results of Diagnostic Tests Based on Enzyme Immunoassay and Cell Culture Cytotoxicity Assay  

PubMed Central

The impact of Clostridium difficile fecal loads on diagnostic test results is poorly understood, but it may have clinical importance. In this study, we investigated the relationship between C. difficile fecal load and the results of four assays: a glutamate dehydrogenase (GDH) enzyme immunoassay (EIA), a toxin A/B antigen EIA (ToxAB), a cell culture cytotoxicity assay (CCA), and PCR targeting the tcdB gene. We also compared the PCR cycle threshold (CT) with the results of quantitative culture using Spearman's rank correlation coefficient. Finally, we sequenced the genomes of 24 strains with different detection profiles. A total of 203 clinical samples harboring toxigenic C. difficile were analyzed and sorted into one of four groups: 17 PCR+ (group 1), 37 PCR+ GDH+ (group 2), 24 PCR+ GDH+ CCA+ (group 3), and 125 PCR+ GDH+ ToxAB+ (group 4). The overall median fecal load in log10 CFU/g was 6.67 (interquartile range [IQR], 5.57 to 7.54). The median fecal bacterial load of groups 1, 2, 3, and 4 were 4.15 (IQR, 3.00 to 4.98), 5.74 (IQR, 4.75 to 6.16), 6.20 (IQR, 5.23 to 6.80), and 7.08 (IQR, 6.35 to 7.83), respectively. Group 1 samples had lower fecal loads than those from each of the other groups (P < 0.001). Group 2 samples had lower fecal loads than those from groups 3 and 4 (P < 0.001). There was a significant correlation between PCR CT and fecal loads (? = ?0.697; P < 0.001). NAP1 strains were associated with the detection of toxins by EIA or CCA (P = 0.041). This study demonstrates an association between C. difficile fecal load and the results of routinely used diagnostic tests.

Dionne, Lea-Laurence; Raymond, Frederic; Corbeil, Jacques; Longtin, Jean; Gervais, Philippe

2013-01-01

55

A Cell Programmable Assay (CPA) chip.  

PubMed

This article describes two kinds of "Cell Programmable Assay" (CPA) chips that utilize passive pumping for the culture and autonomous staining of cells to simply common protocols. One is a single timer channel CPA (sCPA) chip that has one timer channel and one main channel containing a cell culture chamber. The sCPA is used to culture and stain cells using Hoechst nuclear staining dye (a 2 step staining process). The other is a dual timer channel CPA (dCPA) chip that has two timer channels and one main channel with a chamber for cell culture. The dCPA is used here to culture, fix, permeablize, and stain cells using DAPI. The additional timer channel of the dCPA chip allows for automation of 3 steps. The CPA chips were successfully evaluated using HEK 293 cells. In addition, we provide a simplified equation for tuning or redesigning CPA chips to meet the needs of a variety of protocols that may require different timings. The equation is easy to use as it only depends upon the dimensions of microchannel and the volume of the reagent drops. The sCPA and dCPA chips can be readily modified to apply to a wide variety of common cell culture methods and procedures. PMID:20614082

Ju, Jongil; Warrick, Jay; Beebe, David J

2010-08-21

56

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

PubMed

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

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

2008-11-01

57

Limitations of the MTT Assay in Cell Viability Testing  

Microsoft Academic Search

Background. The MTT assay is widely recommended for examining the cytotoxic effect of xenobiotics, assessing proliferation rates, and analyzing cell activity. The aim of this study was to evaluate the usefulness of the MTT assay in examining human lymphocyte viability in own cell culture systems which contained fluphenazine (FPh), a suspected cancer chemopreventive agent, and doxorubicin (DOX), an anticancer drug,

Silesian Piasts

2008-01-01

58

Sequence Homologies between Mycoplasma and Chlamydia spp. Lead to False-Positive Results in Chlamydial Cell Cultures Tested for Mycoplasma Contamination with a Commercial PCR Assay?  

PubMed Central

Mycoplasma contamination is a frequent problem in chlamydial cell culture. After obtaining contradictory contamination results, we compared three commercial PCR kits for mycoplasma detection. One kit signaled contamination in mycoplasma-free Chlamydia pneumoniae cultures. Sequencing of cloned PCR products revealed primer homology with the chlamydial genome as the basis of this false-positive result.

Maass, Viola; Kern, Jan Marco; Poeckl, Matthias; Maass, Matthias

2011-01-01

59

Optimizing stem cell culture.  

PubMed

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

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

2010-11-01

60

Optimizing stem cell culture  

PubMed Central

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

Van Der Sanden, Boudewijn; Dhobb, Mehdi; Berger, Francois; Wion, Didier

2010-01-01

61

Comet assay to measure DNA damage in apoptotic cells  

Microsoft Academic Search

We have used microelectrophoresis technique to study DNA fragmentation in tumor cells undergoing apoptosis induced by NK cells through ADCC. The DNA damage in target cells is proportionate to the time of co-culture with the effector cells. The assay is simple, rapid and inexpensive.

P. Gopalakrishna; Ashok Khar

1995-01-01

62

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

63

Integrated microfluidic devices for combinatorial cell-based assays  

PubMed Central

The development of miniaturized cell culture platforms for performing parallel cultures and combinatorial assays is important in cell biology from the single-cell level to the system level. In this paper we developed an integrated microfluidic cell-culture platform, Cell-microChip (Cell-?Chip), for parallel analyses of the effects of microenvir-onmental cues (i.e., culture scaffolds) on different mammalian cells and their cellular responses to external stimuli. As a model study, we demonstrated the ability of culturing and assaying several mammalian cells, such as NIH 3T3 fibro-blast, B16 melanoma and HeLa cell lines, in a parallel way. For functional assays, first we tested drug-induced apoptotic responses from different cell lines. As a second functional assay, we performed "on-chip" transfection of a reporter gene encoding an enhanced green fluorescent protein (EGFP) followed by live-cell imaging of transcriptional activation of cyclooxygenase 2 (Cox-2) expression. Collectively, our Cell-?Chip approach demonstrated the capability to carry out parallel operations and the potential to further integrate advanced functions and applications in the broader space of combinatorial chemistry and biology.

Yu, Zeta Tak For; Kamei, Ken-ichiro; Takahashi, Hiroko; Shu, Chengyi Jenny; Wang, Xiaopu; He, George Wenfu; Silverman, Robert

2010-01-01

64

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

65

Micronucleus assay in human cells: lymphocytes and buccal cells.  

PubMed

The micronucleus (MN) assay, applied in different surrogate tissues, is one of the best validated cytogenetic techniques for evaluating chromosomal damage in humans. The cytokinesis-block micronucleus cytome assay (CBMNcyt) in peripheral blood lymphocytes is the most frequent method in biomonitoring human populations to evaluate exposure to genotoxic agents, micronutrient deficiency, or excess and genetic instability. Furthermore recent scientific evidence suggests an association between an increased MN frequency in lymphocytes and risk of cancer and other age-related degenerative diseases. The micronucleus cytome assay applied in buccal exfoliated cells (BMNCyt) provides a complementary method for measuring DNA damage and cytotoxic effects in an easily accessible tissue not requiring in vitro culture. The protocol for CBMNcyt described here refers to the use of ex vivo whole blood involving 72 h of culture with the block of cytokinesis at 44 h. BMNCyt protocol reports the established method for sample processing, slide preparation, and scoring. PMID:23896878

Bolognesi, Claudia; Fenech, Michael

2013-01-01

66

Ligand Accumulation in Autocrine Cell Cultures  

SciTech Connect

Cell-culture assays are routinely used to analyze autocrine signaling systems, but quantitative experiments are rarely possible. To enable the quantitative design and analysis of experiments with autocrine cells, we develop a biophysical theory of ligand accumulation in cell-culture assays. Our theory predicts the ligand concentration as a function of time and measurable parameters of autocrine cells and cell-culture experiments. The key step of our analysis is the derivation of the survival probability of a single ligand released from the surface of an autocrine cell. An expression for this probability is derived using the boundary homogenization approach and tested by stochastic simulations. We use this expression in the integral balance equations, from which we find the Laplace transform of the ligand concentration. We demonstrate how the theory works by analyzing the autocrine epidermal growth factor receptor system and discuss the extension of our methods to other experiments with cultured autocrine cells.

Monine, Michael I.; Berezhkovskii, Alexander M.; Joslin, Elizabeth J.; Wiley, H S.; Lauffenburger, Douglas A.; Shvartsman, Stanislav

2005-04-01

67

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

PubMed

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

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

2013-04-01

68

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

PubMed Central

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

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

2012-01-01

69

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

PubMed Central

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

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

2014-01-01

70

Mammalian Cell Culture Simplified.  

ERIC Educational Resources Information Center

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

Moss, Robert; Solomon, Sondra

1991-01-01

71

Keratinocyte-based cell assays: their potential pitfalls.  

PubMed

As an in vitro model system, patient-derived epidermolysis bullosa simplex keratinocytes have had an immense impact on what we know today about keratin filament function and their role in disease development. In the absence of gene therapy, screening compound libraries for new or better drugs is another approach to improve existing treatments for genodermatoses. However in this study, we report of the potential pitfalls when using this type of cell lines as a "reporter" system. When cell lines with different genetic backgrounds are being used in cell-based assays, the greatest obstacle is to determine the most appropriate culture conditions (i.e., the composition of medium, number of cells plated and number of days in culture). We demonstrate how culture conditions can greatly interfere with the cellular response in cell-based assays (cell proliferation, metabolic activity and migration), potentially also giving rise to misleading data. PMID:22983161

Zupancic, Tina; Ozir, Mateja; Törmä, Hans; Komel, Radovan; Liovic, Mirjana

2012-11-01

72

Screening Hybridoma Culture Supernatants Using SolidPhase Radiobinding Assay  

Microsoft Academic Search

\\u000a A large number of hybridoma culture supernatants (up to 200) need to be screened for antibodies at one time. The assay must\\u000a be reliable so that it can accurately identify positive lines, and it must be relatively quick so that the positive lines,\\u000a which are 75–100% confluent, can be fed and expanded as soon as possible after the assay results

Mark Page; Robin Thorpe

73

Screening Hybridoma Culture Supernatants Using SolidPhase Radiobinding Assay  

Microsoft Academic Search

\\u000a A large number of hybridoma culture supernatants (up to 200) need to be screened for antibodies at one time. The assay must\\u000a be reliable so that it can accurately identify positive lines, and it must be relatively quick so that the positive lines,\\u000a which are 75-100% confluent, can be fed and expanded as soon as possible after the assay results

Mark Page; Robin Thorpe

74

Mutation assays involving blood cells that metabolize toxic substances  

DOEpatents

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

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

1999-01-01

75

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

76

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.

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

2013-01-01

77

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

78

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

PubMed Central

A method for assaying neutralizing antibody against variola virus was established by focus counting on HeLa cell cultures. The ND50 titre, i.e., the serum dilution endpoint to give a 50% reduction in the number of foci, was determined with excellent reproducibility. Groups of students 19-20 years of age were revaccinated by the multiple pressure method with serial 10-fold dilutions of a smallpox vaccine and their neutralizing antibody response was assayed by the focus counting assay system and was related to the local skin reactions on the seventh day after inoculation and to the potency of the vaccine administered. There was a significant rise in the antibody level even after inoculation with a vaccine whose potency was as low as 1.3 × 105 pock-forming units/ml. In general, the rise in the log antibody level was proportional to the diameter of the reddening, but a significant rise was found among individuals who had no detectable skin reaction. The skin reaction was greater among individuals with a lower initial antibody level when the vaccine administered had a potency lower than 1.3 × 106 pock-forming units/ml.

Kitamura, Takashi; Shinjo, Nagashige

1972-01-01

79

Microfluidic System for Automated Cell-based Assays  

PubMed Central

Microfluidic cell culture is a promising technology for applications in the drug screening industry. Key benefits include improved biological function, higher quality cell-based data, reduced reagent consumption, and lower cost. In this work, we demonstrate how a microfluidic cell culture design was adapted to be compatible with the standard 96-well plate format. Key design features include the elimination of tubing and connectors, the ability to maintain long term continuous perfusion cell culture using a passive gravity driven pump, and direct analysis on the outlet wells of the microfluidic plate. A single microfluidic culture plate contained 8 independent flow units, each with 104 cells at a flow rate of 50 ?l/day (6 minute residence time). The cytotoxicity of the anti-cancer drug etoposide was measured on HeLa cells cultured in this format, using a commercial lactate dehydrogenase (LDH) plate reader assay. The integration of microfluidic cell culture methods with commercial automation capabilities offers an exciting opportunity for improved cell-based screening.

Lee, Philip J; Ghorashian, Navid; Gaige, Terry A; Hung, Paul J

2007-01-01

80

A Biochemical Assay for Acetylcholinesterase Activity in PC12 Cells  

NSDL National Science Digital Library

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

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

2007-07-10

81

Human Cell Chips: Adapting DNA Microarray Spotting Technology to Cell-Based Imaging Assays  

Microsoft Academic Search

Here we describe human spotted cell chips, a technology for determining cellular state across arrays of cells subjected to chemical or genetic perturbation. Cells are grown and treated under standard tissue culture conditions before being fixed and printed onto replicate glass slides, effectively decoupling the experimental conditions from the assay technique. Each slide is then probed using immunofluorescence or other

Traver Hart; Alice Zhao; Ankit Garg; Swetha Bolusani; Edward M. Marcotte; Joanna Mary Bridger

2009-01-01

82

Measurement of Reactive Oxygen Species in the Culture Media Using Acridan Lumigen PS-3 Assay  

PubMed Central

Reactive oxygen species (ROS) are generated continuously during aerobic metabolism. ROS are highly reactive molecules and in excessive amounts, can lead to protein and DNA oxidation, protein cross-linking, and cell death. Cell-culture models provide a valuable tool in understanding the mechanisms that lead to cell death. Accumulation of ROS within cells and/or their release into the culture media are highly cell type-specific. The ability to estimate ROS levels in the culture media is an important step in understanding the mechanisms contributing to disease processes. In this paper, we describe the optimization of a simple method to estimate ROS levels in the culture media using the Acridan Lumigen PS-3 reagent provided in the Amersham ECL Plus kit (GE Healthcare, UK). We have shown that the Acridan Lumigen PS-3 assay generates ROS-specific chemiluminescence in fresh as well as media stored at ?20°C, in as little as 10–20 ?l of samples. The method was able to detect the dose (of stimulants)- and time (acute and chronic)-dependent changes in ROS levels in media collected from various cell types. Our results suggest that the kit reagents, PBS buffer, and various media did not contribute significantly to the overall chemiluminescence generated in the assay; however, we suggest that the unused medium specific for each cell type should be used as blanks and final readings of test samples normalized against these readings. As this method uses commonly available laboratory equipment and commercially available reagents, we believe this assay is convenient, economical, and specific in estimating ROS released extracellularly into the culture media.

Uy, Benedict; McGlashan, Susan R.; Shaikh, Shamim B.

2011-01-01

83

Method for measuring neurotoxicity of aggregating polypeptides with the MTT assay on differentiated neuroblastoma cells  

Microsoft Academic Search

Reliable in vitro assays are essential for study of the effects of neurotoxic compounds such as ?-amyloid peptides (A?). The MTT assay has been used in cultures of different cells, e.g. SH-SY5Y neuroblastoma cells, for the quantitative measurement of A? toxicity. In our laboratory differentiated SH-SY5Y cells were used in the MTT assay. Cell differentiation with 10?M all-trans-retinoic acid resulted

Zsolt Datki; Anna Juhász; Márta Gálfi; Katalin Soós; Rita Papp; Dénes Zádori; Botond Penke

2003-01-01

84

Stem cell assays in the evaluation of myelotoxicity  

PubMed Central

The concept and characteristics of different types of hematopoietic cells have been described. Hematopoietic stem cells are currently considered to exist in a variety of populations with different degrees of commitment towards a particular cell line. By a combination of animal studies and studies with hematopoietic disorders, the concept of dividing hematopoietic stem cells into uncommitted and committed types has emerged in the past several years. Uncommitted stem cells are capable of differentiation, under the proper stimulus, into either of the cell lines of the hematopoietic system. These cells form a resting population of cells with a low mitoitic rate and a long resting (G0) phase. The committed stem cells are partially differentiated and mature only into one type of cell. The committed stem cell population is relatively more active than the population of uncommitted stem cells. A variety of assays both in vivo and in vitro are currently available for the study of different hematopoietic stem cells. These assays are semiquantitative. The number of colonies of mature cells which develop after the infusion or plating of a population of cells containing the stem cells is proportional to the total number of cells infused. A variety of toxic as well as biological substances have been assayed in these systems and a quantitative depression of the number of colonies produced has been noted by a variety of workers. The degree of depression in the number of colonies varies with the agent in use and the type of assay employed. These studies have demonstrated that toxicity of chemicals on the hematopoietic stem cells can be studied with these in vitro and animal studies to give an assessment of their potential toxicity in the intact organism. The recent development of the Dexter two-layer liquid culture system has provided a new impetus to the research on the uncommitted stem cell in a variety of organisms.

Mangalik, Aroop; Robinson, William A.

1981-01-01

85

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

2014-01-01

86

Novel derivatives of 1,3,4-oxadiazoles are potent mitostatic agents featuring strong microtubule depolymerizing activity in the sea urchin embryo and cell culture assays  

Microsoft Academic Search

A series of novel 1,3,4-oxadiazole derivatives based on structural and electronic overlap with combretastatins have been designed and synthesized. Initially, we tested all new compounds in vivo using the phenotypic sea urchin embryo assay to yield a number of agents with anti-proliferative, anti-mitotic, and microtubule destabilizing activities. The experimental data led to identification of 1,3,4-oxadiazole derivatives with isothiazole (5–8) and

Alex S. Kiselyov; Marina N. Semenova; Natalya B. Chernyshova; Andrei Leitao; Alexandr V. Samet; Konstantine A. Kislyi; Mikhail M. Raihstat; Tudor Oprea; Heiko Lemcke; Margaréta Lantow; Dieter G. Weiss; Nazli N. Ikizalp; Sergei A. Kuznetsov; Victor V. Semenov

2010-01-01

87

Relative embryotoxic potency of p-substituted phenols in the embryonic stem cell test (EST) and comparison to their toxic potency in vivo and in the whole embryo culture (WEC) assay.  

PubMed

The applicability of the embryonic stem cell test (EST) as an alternative for in vivo embryotoxicity testing was evaluated for a series of five p-substituted phenols. To this purpose, the potency ranking for this class of compounds derived from the inhibition of cardiomyocyte differentiation in the EST was compared to in vivo embryotoxic potency data obtained from literature and to the potency ranking defined in the in vitro whole embryo culture (WEC) assay. From the results obtained it appears that the EST was able to identify the embryotoxic potential for p-substituted phenols, providing an identical potency ranking compared to the WEC assay. However, the EST was not able to predict an accurate ranking for the phenols compared to their potency observed in vivo. Only phenol, the least potent compound within this series, was correctly ranked. Furthermore, p-mercaptophenol was correctly identified as a relative potent congener of the phenols tested, but its ranking was distorted by p-heptyloxyphenol, of which the toxicity was overestimated in the EST. It is concluded that when attempting to explain the observed disparity in potency rankings between in vitro and in vivo embryotoxicity, the in vitro models should be combined with a kinetic model describing in vivo absorption, distribution, metabolism and excretion processes of the compounds. PMID:22820428

Strikwold, Marije; Woutersen, Ruud A; Spenkelink, Bert; Punt, Ans; Rietjens, Ivonne M C M

2012-09-01

88

Evaluation of the alamarblue assay for adherent cell irradiation experiments.  

PubMed

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

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

2014-05-01

89

Quantitative Representation of Three-Dimensional Cell Culture Models.  

National Technical Information Service (NTIS)

Three-dimensional mammary cell culture models offer new opportunities for the development of computational techniques for segmentation, localization, and multicellular organization. Under normal conditions, these assays form a symmetrical, hollow structur...

B. Parvin C. Park H. Chang

2007-01-01

90

Human Primary Lung Endothelial Cells in Culture  

PubMed Central

Pulmonary endothelial functions are critical to maintain the low pressure of the pulmonary circulation and effective diffusion capacity of the lung. To investigate pulmonary endothelial cell biology in healthy or diseased lungs, we developed methods to harvest and culture pure populations of primary pulmonary arterial endothelial cells and microvascular endothelial cells from human lung explanted at time of transplantation or from donor lungs not used in transplantation. The purity and characteristics of cultured endothelial cells is ascertained by morphologic criteria using phase contrast and electron microscopy; phenotypic expression profile for endothelial specific proteins such as endothelial nitric oxide synthase, platelet/endothelial cell adhesion molecule, and von Willbrand factor; and endothelial function assays such as Dil-acetylated low-density lipoprotein uptake and tube formation. This detailed method provides researchers with the ability to establish cells for molecular, genetic, and biochemical investigation of human pulmonary vascular diseases.

Xu, Weiling; Mavrakis, Lori; Aldred, Micheala A.; Asosingh, Kewal; Erzurum, Serpil C.

2012-01-01

91

Cell death pattern of a varicose vein organ culture model.  

PubMed

The study aimed to investigate the viability of a varicose vein (VV) organ culture model by assessing cell death pattern. To assess pattern of cell death with time, VV organ cultures were incubated for up to 14 days with regular medium changed. To assess viability, cell death of VV organ cultures treated with sodium azide and their untreated counterparts was assayed. Increased cell death was measured in VV organ cultures from day 0 to 2. Cell death decreased gradually after day 2 and plateaued from day 8 to 14.VV organ cultures treated with sodium azide demonstrated significantly more cell death in tissue (P = 0.001). Cell death measured in cultures treated with sodium azide continued to increase until day 7. In conclusion, this study demonstrated the viability of a VV organ culture model with most cell death occurred within the first two days and then declined to a relatively low level. PMID:23526103

Lim, Chung S; Kiriakidis, Serafim; Paleolog, Ewa M; Davies, Alun H

2013-06-01

92

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

93

Isolation and Culture of Epithelial Stem Cells  

PubMed Central

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

Nowak, Jonathan A.; Fuchs, Elaine

2009-01-01

94

Fermentation with immobilized cell cultures.  

PubMed

For the production of monoclonal antibodies and complex recombinant human proteins or glycoproteins a number of immobilized cell culture systems have been developed. The advantages of such cell culture systems are that cells can be kept in small volumes of cell culture fluid and media can be changed continuously if necessary for induction of product synthesis or removal and harvest of metabolic products. Whereas the hollow fiber and the opticell culture systems can be limited in scaling up the microcarrier system, the fluidized bed bioreactor and the solid bed bioreactor are suitable for scaling up. In contrast to the other systems, the solid bed bioreactor requires no special manipulation for anchoring the cells to the wire springs. In situ cleaning is possible and the beads are reusable. With this cell culture fermentation system, production processes for interferon beta, monoclonal antibodies for interferon alfa and recombinant human tissue plasminogen activator were developed. PMID:3285839

Werner, R G; Merk, W; Walz, F

1988-02-01

95

Polyester ?-assay chip for stem cell studies  

PubMed Central

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

Piraino, Francesco; Selimovic, Seila; Adamo, Marco; Pero, Alessandro; Manoucheri, Sam; Bok Kim, Sang; Demarchi, Danilo; Khademhosseini, Ali

2012-01-01

96

Polyester ?-assay chip for stem cell studies.  

PubMed

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

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

2012-01-01

97

Detection of anaerobic mycoplasmas in cell cultures  

Microsoft Academic Search

Summary  A commercially available anaerobic generator and incubation system that develops a low oxidation-reduction potential was used\\u000a for the assay of cell cultures for mycoplasmal contamination. Mycoplasma broth and agar media supplemented with dextrose,\\u000a yeast extract, and horse serum were used. This system supported growth of some mycoplasmas that failed to grow in incubators\\u000a with 5% CO2 in nitrogen previously used

Gerard J. McGarrity; Lewis L. Coriell

1973-01-01

98

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

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

1974-01-01

99

Plaque assay of rickettsiae in a mammalian cell line.  

PubMed

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. PMID:4208640

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

1974-06-01

100

Canine coronavirus induces apoptosis in cultured cells.  

PubMed

Canine coronavirus (CCoV) is widespread in dogs in several countries and causes mild enteric illness evolving to severe enteritis in young pups. In in vitro cultures canine coronaviruses generally induce extensive cell death, however nature of the events leading to cell death remains largely unknown. We analysed the induction of cytopathic effect by CCoV in a canine fibrosarcoma cell line (A-72) in order to characterize the apoptotic effect in homologous cell system. Following CCoV infection A-72 cell line, which is permissive to CCoV, showed reduced growth rate, as detected by MTT assay, a standard colorimetric assay for measuring cellular proliferation, and underwent to apoptotic death. Starting from 24h after CCoV infection, cells morphology appeared dramatically changed, with cells rounding and detachment from culture surface. Morphologic and biochemical features of apoptosis, such as blebbing of the plasma membrane, translocation of phosphatidilserine to cell surface and annexin V positive staining, nuclear fragmentation, apoptotic bodies formation and DNA laddering, were detected in CCoV-infected cells. Propidium iodide staining of infected culture indicated the appearance of hypodiploid DNA peak corresponding to apoptotic cell population. Commonly to other animal coronavirus infection caspase-3 is likely to contribute to the execution phase of apoptosis induced by CCoV in A-72 cells since we found activation of enzymatic activity as well as procaspase-3 activating cleavage. Apoptotic death of infected cells is detrimental as it causes cell and tissue destruction as well as inflammatory responses. Therefore in the case of CCoV associated gastroenteritis, apoptosis of epithelial mucosa cells may be responsible for pathology induced by CCoV infection. PMID:17254720

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

2007-03-31

101

Ureaplasma infection of cell cultures.  

PubMed Central

Studies were performed to characterize the effects of ureaplasmas in HeLa, 3T6, and CV-1 cell cultures. The ureaplasmas studied were human Ureaplasma urealyticum T960 (serotype VIII), bovine U. diversum T95, simian strain T167-2, ovine strain 1202, canine strain D1M-C, and feline strains 382 and FT2-B. FT2-B was the only ureaplasma to grow in the cell free culture medium, Dulbecco modified Eagle-Earle medium containing 10% fetal bovine serum. The growth pattern of the ureaplasmas varied in the different cell cultures, but each strain grew in at least two of the cell cultures, suggesting a requirement for a product of the cell culture and for low concentrations of urea. When growth occurred, organisms grew to concentrations that approached, but did not equal, those observed in 10B broth. Most, but not all, ureaplasmas grew quickly, reaching peak titers 2 days after infection. Canine strain D1M-C did not grow in 3T6, but showed rapid growth in HeLa and CV-1 cells, killing both cultures, In some systems, e.g., U. urealyticum T960 and simian strain T167-2, the infection persisted, and ureaplasmas could be recovered from cell cultures four passages after infection, when studies were terminated. The cell culture ureaplasmas grew on T agar, but not on mycoplasma agar medium. Images

Kotani, H; McGarrity, G J

1986-01-01

102

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity  

PubMed Central

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with ?III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling ?III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

Anderl, Janet L; Redpath, Stella; Ball, Andrew J

2009-01-01

103

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

104

Adult human endothelial cell enzymatic harvesting. Estimates of efficiency and comparison of crude and partially purified bacterial collagenase preparations by replicate microwell culture and fibronectin degradation measured by enzyme-linked immunosorbent assay.  

PubMed

Reliable enzymatic endothelial cell (EC) harvest methods are required for clinical EC seeding of vascular prostheses by methods analogous to those demonstrated in dogs. But crude collagenases used for EC harvest vary in efficacy and cytotoxicity, and purified collagenases reportedly give low EC yields. To compare different harvest methods, we studied growth curves of primary adult human saphenous vein EC (HSVEC) harvests plated in replicate microwell cultures. The EC yield, defined as attachment-capable ECs obtained per square centimeter of vein lumen, was estimated from the lowest number of ECs counted in lag phase before exponential growth began. With the use of morphometric studies of HSVs that were perfusion-fixed at their original dimensions, the baseline in situ density of ECs available for harvest from HSV was estimated at 1.3 X 10(5) EC/cm2. Crude (CBC) and partially purified bacterial collagenase (PBC) solutions at concentrations with equal levels of basement membrane lysis activity (BMLA) were compared by the replicate microwell method in a series of 21 harvests (six CBC, eight PBC, and seven enzyme-free control harvests). All 14 enzymatic harvests produced confluent EC cultures with no significant difference in mean harvest efficiency between CBC (12% of in situ EC number) and PBC (15%). However, PBC caused less degradation of human fibronectin (p less than 0.0001) as measured by an enzyme-linked immunosorbent assay employing a fibronectin-specific monoclonal antibody. These data suggest that chemically defined mixtures of pure enzymes with BMLA equal to the BMLA of crude collagenase might allow reliable EC harvesting without sacrifice in EC yield but with improved preservation of structures at the EC periphery. EC losses during initial vein dissection may have contributed to the low 12% to 15% efficiency we observed. PMID:3023710

Sharefkin, J B; Van Wart, H E; Cruess, D F; Albus, R A; Levine, E M

1986-12-01

105

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.

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

2010-01-01

106

Deltamethrin induces apoptotic cell death in cultured cerebral cortical neurons  

Microsoft Academic Search

In this study we investigated the induction of apoptotic cell death and its potential mechanisms in cultured cortical neurons in response to deltamethrin exposure. The cultured cortical neurons were treated at 7 days with deltamethrin at concentrations of 10, 100, and 1000 nM, respectively. MTT assay showed that higher concentrations of deltamethrin (100 and 1000 nM) decreased neuronal viability in

Aiguo Wu; Long Li; Yugu Liu

2003-01-01

107

Suspension culture of mammalian cells.  

PubMed

Mammalian cell suspension culture systems are being used increasingly in the biotechnology industry. This is due to their many advantages including simplicity and homogeneity of culture. Suspension systems are very adaptable (e.g., for microcarrier, microencapsulation, or other methods of culture). Their engineering is thoroughly understood and standardized at large scale, and automation and cleaning procedures are well established. Suspension systems offer the possibility of quick implementation of production protocols due to their ability to be scaled easily once the basic culture parameters are understood. The only main disadvantage of the suspension culture systems to date is their inapplicability for the production of human vaccines from either primary cell lines or from normal human diploid cell lines (Hayflick et al., 1987 and references therein). One of the great advantages of suspension culture is the opportunity it provides to study interactions of metabolic and production phenomena in chemostat or turbidostat steady-state systems. Furthermore, in suspension culture systems from which cell number and cell mass measurements are easy to obtain, rigorous and quantitative estimations of the effects of growth conditions or perturbations of metabolic homeostasis can be made. Such studies can speed up the development of optimal processes. With our increasing understanding of factors influencing expression in mammalian cells (Cohen and Levinson, 1988; Santoro et al., 1988) and the direct application of new methods in suspension culture (Rhodes and Birch, 1988), its usefulness and importance is likely to increase in the future. In this chapter, we have described some of the potential uses of the various suspension culture systems and have covered most of the established technology and literature. Due to the rapid developments and needs in the biotechnology industry and the versatility of suspension culture systems, it is probable that many more variations on this theme will evolve in the near future at both the pilot and production scales. PMID:1367062

Birch, J R; Arathoon, R

1990-01-01

108

Aflatoxins in Mammalian Cell Cultures.  

National Technical Information Service (NTIS)

KB and HeLa mammalian tissue culture cells were cultured in the presence of 0.4, 1, and 4 ppm of aflatoxin. The incorporation of labeled uridine into the different RNA components separated by sucrose gradient ultracentrifugation and by methylated albumin ...

R. A. Chung

1968-01-01

109

Enhanced Cell Culture Growth Rates.  

National Technical Information Service (NTIS)

Major project tasks included assembly of an ultrasonic treatment array; measurement of the cell culture growth rate as a function of media concentration, ultrasonic frequency, and ultrasonic power level and dosage; and evaluation of some physiological ind...

S. R. Taylor

1988-01-01

110

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

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; Weissmann, Charles

2012-05-01

111

Assays for Endogenous and Exogenous Lymphoid Leukosis Viruses and Chick Helper Factor with RSV(-) Cell Lines  

PubMed Central

Japanese quail cells transformed by the envelope-defective Bryan high-titer strain of Rous sarcoma virus [R(?)Q] were used as a source of the Rous sarcoma virus genome in three kinds of assays. (i) The simplest and most sensitive assay for infectious, endogenous viruses of the chicken belonging to subgroup E involved infection of a mixture of R(?)Q cells and turkey cells with the sample and assay of supernatants of these cells for focus formation on subgroup E susceptible cells. (ii) Inactivated Sendai virus-induced fusion of R(?)Q cells with live test cells was found to be a specific method for detection of chick helper factor. Focus formation by supernatant of the fused cells on subgroup E susceptible cells was correlated with the presence of subgroup E envelope glycoprotein on the plasma membranes of test cells. Whole blood cells as well as fibroblasts could be used in this assay. (iii) A method of assay for exogenous lymphoid leukosis viruses in which mixed cultures of R(?)Q cells and C/E cells and assay of supernatants for focus formation on C/E cells was as sensitive as assays presently used for exogenous lymphoid leukosis virus. Because no infectious Rous sarcoma virus was used as part of the procedure, the assays for infectious virus described here yielded pure pseudotypes of the input virus, an advantage for determining purity and subgroup of the input virus.

Crittenden, L. B.; Eagen, D. A.; Gulvas, F. A.

1979-01-01

112

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

113

A Comparison of Colorimetric and DNA Quantification Assays for the Assessment of Meniscal Fibrochondrocyte Proliferation in Microcarrier Culture  

Microsoft Academic Search

We have developed and refined a rapid, reliable method for the evaluation of attachment and proliferation of ovine meniscal chondrocytes in microcarrier culture. Assays measuring both mitochondrial activity, using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and MTS [3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium], and DNA synthesis with a PicoGreen assay were compared. The MTT assay was the most sensitive at lower cell concentrations and enabled accurate assessment

Moreica B. Pabbruwe; Karina Stewart; Julian B. Chaudhuri

2005-01-01

114

Chlorophyllous totipotent maize cell cultures  

US Patent & Trademark Office Database

The subject invention provides totipotent, chlorophyllous, cell cultures of maize. In addition, the methods of producing such cultures are applicable to other related species, including cereals such as rice, oats, barley, and heat. The subject cultures are valuable for herbicide studies, studies for enhancing photosynthesis, and genetic manipulation, such as plastid transformation. The methods of the subject invention are capable of providing high percentages of totipotent cells. These cells are capable of sustained cell division and are competent for regeneration over long periods; they provide high-quality target tissue for nuclear and organelle transformation. The invention also describes methods for the introduction of heterologous DNA into the chloroplast genome. The present invention also provides methods, vectors, and gene constructs for enhancing expression of a recombinant nucleic acid sequence in transgenic plants and plant tissues.

2011-10-18

115

Effect of lipoproteins on cultured human mesangial cells  

Microsoft Academic Search

It was recently reported that low-density lipoprotein (LDL) promotes mesangial cell proliferation, and oxidized LDL is cytotoxic for mesangial cells. However, there have been few studies about the effects of other lipoproteins on mesangial cells. Accordingly, we investigated the effect of various lipoproteins on cultured human mesangial cells using 3H-thymidine (3H-TdR) incorporation and cell counting assays. We also investigated the

Yoji Nishida; Noriaki Yorioka; Hiroaki Oda; Michio Yamakido

1997-01-01

116

Improved FSH sensitisation and aromatase assay in human granulosa-lutein cells  

Microsoft Academic Search

Granulosa-lutein (GL) cells from follicular aspirates from women undergoing in-vitro fertilization (IVF) treatment are usually refractory to follicle stimulating hormone (FSH) regarding the induction and\\/or maintenance of aromatase activity which converts androgens (e.g. testosterone) to oestrogens. The normal method of assaying FSH-stimulated aromatase activity in GL cell cultures is to add exogenous testosterone throughout the cell culture period and measure

A. Lambert; S. D. Harris; P. Knaggs; W. R. Robertson

2000-01-01

117

ES cell-derived neuroepithelial cell cultures.  

PubMed

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells. Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5). PMID:18704173

Karki, Shreeya; Pruszak, Jan; Isacson, Ole; Sonntag, Kai C

2006-12-01

118

Azo dyes and carcinogenic aromatic amines in cell cultures  

Microsoft Academic Search

Azo dyes are widely used in the food, pharmaceutical, cosmetic, textile and leather industries. They can be reduced by azoreductases in intestinal bacteria, liver cells and skin surface microflora so that aromatic amines are released. In this study an analytical system for the determination of carcinogenic aromatic amines at the picogram to femtogram level and a cell culture assay to

S. Hildenbrand; F. W. Schmahl; R. Wodarz; R. Kimmel; P. C. Dartsch

1999-01-01

119

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

120

Cultured Human Renal Cortical Cells  

NASA Technical Reports Server (NTRS)

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

1998-01-01

121

Shape memory polymers for active cell culture.  

PubMed

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

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

2011-01-01

122

Mesenchymal stem cells from patients to assay bone graft substitutes.  

PubMed

Bio-engineered scaffolds used in orthopedic clinical applications induce different tissue responses after implantation. In this study, non-stoichiometric Mg(2+) ions and stoichiometric apatites, which are used in orthopedic surgery as bone substitutes, have been assayed in vitro with human adult mesenchymal stem cells (hMSC) to evaluate cytocompatibility and osteoconductivity. hMSCs from the bone marrow aspirates of orthopedic patients were isolated and analyzed by flow cytometry for the surface markers Stro1, CD29, CD44, CD71, CD73, CD90, CD105 (positive) and CD45, CD235 (negative). The hMSC were analyzed for self-renewal capacity and for differentiation potential. The hMSC, which were grown on different biomaterials, were analyzed for (i) cytotoxicity by AlamarBlue metabolic assay, (ii) osteoconductivity by ELISA for activated focal adhesion kinase, (iii) cytoskeleton organization by fluorescence microscopy, and (iv) cell morphology which was investigated by scan electron microscopy (SEM). Results indicate that isolated cell populations agree with minimal criteria for defining hMSC cultures. Non-stoichiometric Mg(2+) and stoichiometric apatites, in granular form, represent a more favorable environment for mesenchymal stem cell adhesion and growth compared to the non-stoichiometric Mg(2+) apatite, in nano-structured paste form. This study indicates that different forms of biomaterials modulate osteoconductivity and cellular growth by differential activation focal adhesion kinase. PMID:23129455

Manfrini, M; Di Bona, C; Canella, A; Lucarelli, E; Pellati, A; D'Agostino, A; Barbanti-Bròdano, G; Tognon, M

2013-06-01

123

Three-Dimensional Co-Culture Assay System for Angiogenesis and Metastasis  

Cancer.gov

This technology features an assay for the detection and measurement of angiogenesis and metastasis. A three-dimensional co-culture system has been developed that closely mimics the in vivo environment in which angiogenesis and metastatic tumors develop.

124

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

PubMed

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

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

2014-07-01

125

Development of a Treatment Algorithm for Streptococci and Enterococci from Positive Blood Cultures Identified with the Verigene Gram-Positive Blood Culture Assay  

PubMed Central

Seventy-eight blood cultures with a Gram stain result of Gram-positive cocci in pairs and/or chains were evaluated with the Nanosphere Verigene Gram-positive blood culture (BC-GP) assay. The overall concordance of the assay with culture was 89.7% (70/78 cultures), allowing for the development of a targeted treatment algorithm.

Alby, Kevin; Daniels, Lindsay M.; Weber, David J.

2013-01-01

126

Variability of acid hydrolase activities in cultured skin fibroblasts and amniotic fluid cells  

Microsoft Academic Search

The specific activities of lysosomal hydrolases in cultured skin fibroblasts and amniotic fluid cells showed wide and unpredictable variations between cultures, which may lead to difficulty in differentiating normal, heterozygous, and homozygous cells. However, the variability for a given culture was similar for all enzymes assayed, so that a clearer differentiation of a relative deficiency of a given enzyme could

E Young; P Willcox; A E Whitfield; A D Patrick

1975-01-01

127

A modified plaque assay and infected cell hybridization assay for wild-type and recombinant LuIII autonomous parvovirus.  

PubMed

We describe a convenient infected cell hybridization assay for determining the infectious titer of a recombinant, replication-defective parvovirus. We previously generated recombinant derivatives of the autonomous parvovirus LuIII, transducing the luciferase reporter gene. Since luciferase expression is not readily detected at the single cell level, and since the recombinants cannot form plaques, we developed an alternative assay for infectious particles. Co-infection of NB324K cells with wild-type LuIII (multiplicity of infection ca. 5) and the recombinant virions allows amplification of the transducing DNA, which can be detected by hybridization with a probe for the reporter gene. Cell lysis and DNA transfer to a nylon membrane is performed in situ, in the culture dish, and hybridization is performed with a digoxigenin-labeled probe, using immunological detection. During this work we developed a conveniently modified plaque assay for wild-type LuIII that should be applicable to other lytic viruses. The modification employs a reduced volume of agarose overlay that is in turn overlaid with liquid medium, thus avoiding the need to maintain stocks of culture medium at higher than normal concentration. PMID:8068342

Maxwell, I H; Maxwell, F

1994-05-01

128

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

129

Cell Cultivation and Sensor-Based Assays for Dynamic Measurements of Cell Vitality  

Microsoft Academic Search

\\u000a \\u000a Cell cultivation\\u000a is a fundamental tool in tissue engineering\\u000a as well as in biomedical research. Choice of cell source and the control of cultivation parameters will determine the biological\\u000a relevance and quality of the results. There are numerous biochemical and cellular assays available to test the vitality\\u000a , i.e. the metabolic\\u000a \\u000a and functional activities\\u000a \\u000a , of cells in culture. Most

Angela M. Otto

130

Trends in cell culture technology.  

PubMed

Dynamic macroscale bioreactor systems are the most recent breakthrough in cell culture technology. This major achievement, at the beginning of the 21st century, fortunately coincided with an embarrassing gap in the measures to predict the safety and modes of action of chemicals, cosmetics, air particles and pharmaceuticals. The major hurdles to the translation of these breakthrough achievements of cell culture technology into meaningful solutions for predictive high throughput substance testing remain miniaturization from the milliliter to the microliter scale and the supply of relevant amounts of standardized human tissue. This chapter provides insights into the latest developments in this area, illustrates an original multi-micro-organ bioreactor concept and identifies highways for closing the gap. PMID:22437811

Marx, Uwe

2012-01-01

131

A simple colony-formation assay in liquid medium, termed 'tadpoling', provides a sensitive measure of Saccharomyces cerevisiae culture viability.  

PubMed

Here we describe the first high-throughput amenable method of quantifying Saccharomyces cerevisiae culture viability. Current high-throughput methods of assessing yeast cell viability, such as flow cytometry and SGA analysis, do not measure the percentage viability of a culture but instead measure cell vitality or colony fitness, respectively. We developed a method, called tadpoling, to quantify the percentage viability of a yeast culture, with the ability to detect as few as one viable cell amongst ~10(8) dead cells. The most important feature of this assay is the exploitation of yeast colony formation in liquid medium. Utilizing a microtiter dish, we are able to observe a range of viability of 100% to 0.0001%. Comparison of tadpoling to the traditional plating method to measure yeast culture viability reveals that, for the majority of Saccharomyces species analyzed there is no significant difference between the two methods. In comparison to flow cytometry using propidium iodide, the high-throughput method of measuring yeast culture viability, tadpoling is much more accurate at culture viabilities?cell viability. PMID:24185677

Welch, Aaron Z; Koshland, Douglas E

2013-12-01

132

A novel coagulation assay incorporating adherent endothelial cells in thromboelastometry.  

PubMed

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

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

2013-05-01

133

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

PubMed Central

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

Huyck, Lynn; Ampe, Christophe

2012-01-01

134

Pure-culture and enzymatic assay for starch-polyethylene degradable plastic biodegradation with Streptomyces species  

Microsoft Academic Search

Eleven starch-polyethylene degradable plastic films were prepared from masterbatches from Archer Daniels Midland Inc. (ADM), EcoStar Inc. (SLS), and Fully Compounded Plastic Inc. The biodegradability of initial and 70°C heat-treated materials was determined using a pure-culture assay withStreptomyces badius 252,S. setonii 75Vi2, orS. viridosporus T7A or without bacterial culture (control). Films were treated with 10-foldS. setonii culture concentrates and compared

Anthony L. Pometto; Kenneth E. Johnson; Meera Kirn

1993-01-01

135

Assessment of environmental contamination associated with a mammalian cell transformation assay  

Microsoft Academic Search

Summary  To estimate worker exposures to, and environmental contamination from, test chemicals and organic solvents used in an in vitro\\u000a assay to assess the carcinogenic potential of chemicals, sodium fluorescein, a noncarcinogenic fluorescent material, was dissolved\\u000a in tissue culture medium used to maintain early passage hamster embryo cells. Personal and environmental samples were taken\\u000a over a 14-d period. The assay was

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

1981-01-01

136

In vitro assays for adhesion and migration of osteoblastic cells (Saos-2) on titanium surfaces.  

PubMed

The first event occurring at the boundary between a metal implant and living tissue is the attachment of cells onto the metal surface of the implant. The attachment characteristics of the metal in this situation are critical in determining its biocompatibility and usefulness as artificial bone and tooth implants. Using the human osteosarcoma cell line Saos-2, we attempted to establish simple and reliable methods for evaluating the attachment of cultured osteoblastic cells onto titanium samples that had been subjected to various surface treatments. Fluorescence actin imaging showed that cells cultured on titanium with hydrofluoric acid etching (HF-Ti) exhibited delayed spreading of their cytoplasm, as compared to cells cultured for the same length of time on nitrided titanium or physically polished titanium. The HF-Ti-cultured cells also exhibited poor assembly of focal contacts, as visualized by vinculin immunofluorescence. Furthermore, in motility assays based on an in vitro wound model, cells cultured on HF-Ti migrated more slowly than cells cultured on other titanium surfaces. These data suggest that Saos-2 cells attach less effectively to the HF-Ti surface. The methods described in this study should be useful for assessing the initial interactions of cultured cells with various materials, including metals. PMID:16450122

Li, Chun-yu; Gao, Shuang-yan; Terashita, Takehiro; Shimokawa, Tetsuya; Kawahara, Haruyuki; Matsuda, Seiji; Kobayashi, Naoto

2006-06-01

137

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

138

Use of a Transplacental Host-Mediated Culture System for Assay of Transformation by Nitroso Compounds.  

National Technical Information Service (NTIS)

Nitroso compounds, pesticides and their nitroso derivatives were tested for the ability to transform hamster cells to potentially tumorigenic cells. Pregnant hamsters were injected intraperitoneally with the chemicals and embryo cell cultures were prepare...

J. M. Quarles C. K. Schenley R. W. Tennant

1975-01-01

139

Immunofluorescent Cell-Counting Assay of Rift Valley Fever Virus.  

National Technical Information Service (NTIS)

Rift Valley fever (RVF) virus was quantitatively assayed by enumerating the cells containing fluorescent viral antigens after infection of L cell monolayers. Efficiency and speed of virus attachment were markedly enhanced when augmented by centrifugal for...

N. Hahon

1968-01-01

140

Assay of Chikungunya Virus in Cell Monolayers by Immunofluorescence.  

National Technical Information Service (NTIS)

Chikungunya virus was assayed quantitatively by counting immunofluorescent foci after infection of BHK21/C13 cell monolayers. The speed and efficiency of virus attachment to cells were markedly enhanced when augmented by centrifugal force. By this procedu...

N. Hahon W. A. Hankins

1969-01-01

141

Correlation of Micronucleus and Apoptosis Assays with Reproductive Cell Death.  

National Technical Information Service (NTIS)

The relationship between ionizing radiation-induced cell killing and DNA damage measured by the micronucleus and apoptosis assays was determined in three established cell lines (L929, HL-60, and Chang). Irradiation experiments revealed a dose-dependent in...

M. Abend A. Rhein K. P. Gilbertz W. F. Blakely D. vanBeuningen

1995-01-01

142

A high-throughput assay of yeast cell lysis for drug discovery and genetic analysis  

Microsoft Academic Search

The identification of new antifungal molecules is an important goal of current anti-infective research. To achieve this goal, alternatives to traditional growth inhibition–based screening have been developed in recent years. In this study, we describe an assay to detect molecules that disrupt yeast cell integrity by using the release of adenylate kinase (AK) into culture medium as a reporter of

Louis DiDone; Thomas Scrimale; Bonnie K Baxter; Damian J Krysan

2010-01-01

143

Cytotoxic activity of extracts from Linum cell cultures.  

PubMed

Callus and suspension cultures of Linum narbonense and Linum leonii were developed to study the production of lignans and their cytotoxic activity. Justicidin B was determined to be the main lignan. The maximal yield of justicidin B up to 2.22 mg/g of the cell dry weight was detected in the callus cultures of L. leonii, followed by the callus cultures of L. narbonense (1.57 mg/g dwt). The cytotoxicity of the obtained extracts was measured using the MTT-dye assay. L. narbonense and L. leonii both showed cytotoxic activity. PMID:15664462

Vasilev, N P; Ionkova, I

2005-01-01

144

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.

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

2009-01-01

145

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

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

1972-01-01

146

Multizone Paper Platform for 3D Cell Cultures  

PubMed Central

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

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

2011-01-01

147

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

148

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

149

Use of the tetrazolium assay in measuring the response of human tumor cells to ionizing radiation  

SciTech Connect

Three human tumor cell lines of widely differing radiosensitivity were used to examine the characteristics of the 3-(4,5-dimethyl(thiazol-2-yl)-3,5-diphery)tetradium bromide (MTT) assay and to select suitable conditions for its use in assessing the response of cells to ionizing radiation. The optimal concentration of MTT and the time of incubation of the cells with MTT were individualized for each cell line. The relationship between absorbance and cell number was not linear over the wide range of cell numbers that were used. A calibration curve of absorbance against cell number for each cell line was therefore used. Using the assay to quantify metabolically viable cells, growth curves of irradiated and unirradiated cells were constructed on days 0-14 after irradiation. Accurate surviving fractions could be calculated only when cells were in exponential growth. Using this modification to its interpretation, the MTT assay was able to provide a reproducible measure of survival, which compared well with clonogenic cell survival measurements. However, the necessity to optimize conditions of the MTT assay for each cell line severely limits its usefulness in determining the radiosensitivity of cells in primary human tumor cultures.

Price, P.; McMillan, T.J. (Institute of Cancer Research, Sutton, Surrey (England))

1990-03-01

150

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

151

The Detection of Pyrogens in Blood Products Using an Ex Vivo Whole Blood Culture Assay  

Microsoft Academic Search

Induction of interleukin-6 (IL-6) secretion by whole blood cultures (WBC) was used as an in vitro assay system for pyrogen-induced inflammatory reactions. The assay system was very sensitive to Eschericia coli (E.coli) endotoxin (< 10 pg\\/ml). The potential pyrogenic effects of human serum albumin (HSA), Fibronectin (Fn) and stabilised human serum (SHS) solutions were analyzed using this system. None of

Edmund J. Pool; Gamieda Johaar; Sandra James; Ignatius Petersen; Patrick Bouic

1998-01-01

152

Differentiation and Development in Plant Cell Cultures.  

National Technical Information Service (NTIS)

Differentiation in plant cell cultures was studied by tissue culture and genetic approaches. The influence of various environmental factors on differentiation was elucidated using Digitalis lanata protoplasts in the first part of the study. The molecular ...

R. Puupponen-Pimiae

1995-01-01

153

Detection of Mycobacterium tuberculosis in BACTEC 12B broth cultures by the Roche Amplicor PCR assay.  

PubMed

To evaluate the ability of the Amplicor MTB Assay to detect Mycobacterium tuberculosis complex (MTBC) organisms in BACTEC 12B broth cultures, 249 cultures with a growth index (GI) of > or = 20 from 160 patients were tested retrospectively. Specimens were processed by standard methods, and then BACTEC 12B vials and Middlebrook 7H11/7H115 plates were inoculated, incubated, and interpreted in accordance with the manufacturer's instructions and laboratory protocol. From 12B vials with a GI of > or = 20, and aliquot of broth was removed and frozen at -20 degrees C until assayed by PCR. PCR results were compared to those obtained by the usual laboratory protocol, whereby MTBC organisms were identified by a DNA probe assay performed on broth from 12B vials with a GI or > or = 300 or on colonies from solid medium. Of the 249 broth cultures evaluated, 142 contained mycobacteria, including 44 that contained MTBC organisms. Of these 44 cultures, 41 were PCR positive; the 3 that were PCR negative were blood specimens collected in an Isolator tube. All 98 cultures with nontuberculous mycobacteria and the 107 that did not contain mycobacteria were PCR negative. Thus, the sensitivity and specificity of PCR were 93 and 100%, respectively. For those culture sin which MTBC organisms were identified by both the DNA probe and PCR assays, the mean time from specimen inoculation to detection and identification of MTBC organisms was 16 (range, 4 to 26) days for the PCR and 28 (range, 13 to 43) days for the DNA probe assay (P < 0.0001). In summary, PCR is a rapid, reliable method for detection of MTBC organisms in BACTEC 12B broth cultures with a GI of > or = 20. PMID:9157150

Smith, M B; Bergmann, J S; Woods, G L

1997-04-01

154

Detection of Mycobacterium tuberculosis in BACTEC 12B broth cultures by the Roche Amplicor PCR assay.  

PubMed Central

To evaluate the ability of the Amplicor MTB Assay to detect Mycobacterium tuberculosis complex (MTBC) organisms in BACTEC 12B broth cultures, 249 cultures with a growth index (GI) of > or = 20 from 160 patients were tested retrospectively. Specimens were processed by standard methods, and then BACTEC 12B vials and Middlebrook 7H11/7H115 plates were inoculated, incubated, and interpreted in accordance with the manufacturer's instructions and laboratory protocol. From 12B vials with a GI of > or = 20, and aliquot of broth was removed and frozen at -20 degrees C until assayed by PCR. PCR results were compared to those obtained by the usual laboratory protocol, whereby MTBC organisms were identified by a DNA probe assay performed on broth from 12B vials with a GI or > or = 300 or on colonies from solid medium. Of the 249 broth cultures evaluated, 142 contained mycobacteria, including 44 that contained MTBC organisms. Of these 44 cultures, 41 were PCR positive; the 3 that were PCR negative were blood specimens collected in an Isolator tube. All 98 cultures with nontuberculous mycobacteria and the 107 that did not contain mycobacteria were PCR negative. Thus, the sensitivity and specificity of PCR were 93 and 100%, respectively. For those culture sin which MTBC organisms were identified by both the DNA probe and PCR assays, the mean time from specimen inoculation to detection and identification of MTBC organisms was 16 (range, 4 to 26) days for the PCR and 28 (range, 13 to 43) days for the DNA probe assay (P < 0.0001). In summary, PCR is a rapid, reliable method for detection of MTBC organisms in BACTEC 12B broth cultures with a GI of > or = 20.

Smith, M B; Bergmann, J S; Woods, G L

1997-01-01

155

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

PubMed Central

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

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

2011-01-01

156

Cigarette Smoke Toxicants Alter Growth and Survival of Cultured Mammalian Cells  

Microsoft Academic Search

Our purpose was to determine the effects of six cigarette toxicants (pyridine, nicotine, 2-ethylpyridine, 3-ethylpyridine, p- cresol, and pyrazine) on three types of cultured mammalian cells (human umbilical vein endothelial cells (HUVECs), human microvascular endothelial cells (HMVECs), and NIH 3T3 cells) using a cell proliferation\\/survival assay. Synchronized cells were cultured in proliferation or survival medium containing various doses (10? 18M-10?

Richard Yu; M. Wu; S. Lin; Prue Talbot

2006-01-01

157

The detection of pyrogens in blood products using an ex vivo whole blood culture assay.  

PubMed

Induction of interleukin-6 (IL-6) secretion by whole blood cultures (WBC) was used as an in vitro assay system for pyrogen-induced inflammatory reactions. The assay system was very sensitive to Eschericia coli (E coli) endotoxin (< 10 pg/ml). The potential pyrogenic effects of human serum albumin (HSA), Fibronectin (Fn) and stabilised human serum (SHS) solutions were analyzed using this system. None of the products assayed had an effect on the sensitivity of the WBC assay. Spike recovery studies with isolated endotoxin, gram positive and gram negative bacteria showed that none of the products had an effect on the spike recovery of these pyrogenic substances. Good correlations were found between the WBC assay and the rabbit assay for pyrogens for all the production batches tested. When these samples were analysed by the limulus amoebocyte lysate (LAL) assay, the LAL test gave anomalous results for 1 out of the 22 production batches tested. This batch gave a false negative result on the LAL assay and might be indicative of the inability of the LAL assay to detect pyrogens other than endotoxin. PMID:9682126

Pool, E J; Johaar, G; James, S; Petersen, I; Bouic, P

1998-01-01

158

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

159

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

PubMed

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

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

2006-03-01

160

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.

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

2006-01-01

161

Replication of cultured lung epithelial cells  

Microsoft Academic Search

The authors have investigated the conditions necessary to support replication of lung type 2 epithelial cells in culture. Cells were isolated from mature fetal rabbit lungs (29d gestation) and cultured on feeder layers of mitotically inactivated 3T3 fibroblasts. The epithelial nature of the cells was demonstrated by indirect immunofluorescent staining for keratin and by polyacid dichrome stain. Ultrastructural examination during

D. Guzowski; R. Bienkowski

1986-01-01

162

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

163

New flow cytometric assays for monitoring cell-mediated cytotoxicity  

PubMed Central

The exact immunologic responses after vaccination that result in effective antitumor immunity have not yet been fully elucidated and the data from ex vivo T-cell assays have not yet defined adequate surrogate markers for clinical efficacy. A more detailed knowledge of the specific immune responses that correlate with positive clinical outcomes should help to develop better or novel strategies to effectively activate the immune system against tumors. Furthermore, clinically relevant material is often limited and, thus, precludes the ability to perform multiple assays. The two main assays currently used to monitor lymphocyte-mediated cytoxicity in cancer patients are the 51Cr-release assay and IFN-? ELISpot assay. The former has a number of disadvantages, including low sensitivity, poor labeling and high spontaneous release of isotope from some tumor target cells. Additional problems with the 51Cr-release assay include difficulty in obtaining autologous tumor targets, and biohazard and disposal problems for the isotope. The ELISpot assays do not directly measure cytotoxic activity and are, therefore, a surrogate marker of cyotoxic capacity of effector T cells. Furthermore, they do not assess cytotoxicity mediated by the production of the TNF family of death ligands by the cytotoxic cells. Therefore, assays that allow for the simultaneous measurement of several parameters may be more advantageous for clinical monitoring. In this respect, multifactor flow cytometry-based assays are a valid addition to the currently available immunologic monitoring assays. Use of these assays will enable detection and enumeration of tumor-specific cytotoxic T lymphocytes and their specific effector functions and any correlations with clinical responses. Comprehensive, multifactor analysis of effector cell responses after vaccination may help to detect factors that determine the success or failure of a vaccine and its immunological potency.

Zaritskaya, Liubov; Shurin, Michael R; Sayers, Thomas J; Malyguine, Anatoli M

2010-01-01

164

Hepatitis B virus infection and replication in primary cultured human granulosa cells  

Microsoft Academic Search

To investigate the infection and replication of hepatitis B virus (HBV) in primary cultured human granulosa cells. Human granulosa\\u000a cells were cultured with HBV-positive serum. Media were collected and assayed for HBsAg and HBeAg by ELISA, and HBV DNA by\\u000a quantitative PCR. HBsAg and HBcAg were detected by immunocytochemistry in cultured cells. HBV DNA and RNA were extracted and\\u000a amplified

Yan JinFeng; Feng Ye; Juanzi Shi; Hongtao Qiu; Yingren Zhao; Shumei Lin; Tianyan Chen; Min Liu; Yingli He; Shulin Zhang

2011-01-01

165

Optimisation of cell-based assays for medium throughput screening of oxidative stress.  

PubMed

Identification and development of cell-based assays adapted to medium throughput screening requirements is important when screening chemicals for their potential toxic properties. We describe here rapid fluorometer-based and spectrophotometer-based microplate assays which allow for the evaluation of oxidative stress in hepatocyte cell cultures by measurement of three markers: production of hydroperoxide assessed by the DCFH-DA probe, cellular antioxidant status by measurement of reduced glutathione and glutathione peroxidase activity and cytotoxicity and mitochondrial damage by evaluation of the mitochondrial transmembrane potential with rhodamine 123 fluorescent dye. The assays described here are rapid, simple and inexpensive, which are desirable when setting up screening assays. This system should be useful in selecting candidate compounds during the pre-development phase of agrochemicals or pharmaceuticals. It should also be of interest for retrospective and explanatory studies of mechanisms underlying toxicity observed in vivo. PMID:12650675

Lautraite, S; Bigot-Lasserre, D; Bars, R; Carmichael, N

2003-04-01

166

Zinc protects against oxidative damage in cultured human retinal pigment epithelial cells  

Microsoft Academic Search

This study was undertaken to determine whether bioavailable zinc can influence the effects of oxidative stress on cultured human retinal pigment epithelial (RPE) cells. RPE cells were maintained for 7 d in culture medium containing 14 ?M total zinc, or in medium containing 0.55 ?M total zinc. After 1 week, MTT assays were performed to determine the relative cytotoxicity of

David J Tate; Michael V Miceli; David A Newsome

1999-01-01

167

In vitro genotoxicity assay of sidestream smoke using a human bronchial epithelial cell line  

Microsoft Academic Search

Genotoxic effects of air contaminants, such as gaseous or particulate compounds, have been difficult to investigate due to inefficient methods for exposing cell cultures directly to these substances. New cultivation and exposure techniques enable treatment of epithelial cells with sample atmospheres with subsequent in vitro assays, as demonstrated by a new system called CULTEX (CULTEX: patent No. DE 19801763; PCT\\/EP99\\/00295),

L. Wolz; G. Krause; G. Scherer; M. Aufderheide; U. Mohr

2002-01-01

168

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

PubMed Central

The comet assay is a simple and cost effective technique, commonly used to analyze and quantify DNA damage in individual cells. The versatility of the comet assay allows introduction of various modifications to the basic technique. The difference in the methylation sensitivity of the isoschizomeric restriction enzymes HpaII and MspI are used to demonstrate the ability of the comet assay to measure the global DNA methylation level of individual cells when using cell cultures. In the experiments described here, a medium-throughput comet assay and methylation sensitive comet assay are combined to produce a methylation sensitive medium-throughput comet assay to measure changes in the global DNA methylation pattern in individual cells under various growth conditions.

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

2014-01-01

169

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

170

A continuous perfusion microplate for cell culture.  

PubMed

We describe a 96-well microplate with fluidically connected wells that enables the continuous fluid perfusion between wells without the need for external pumping. A single unit in such a perfusion microplate consists of three wells: a source well, a sample (cell culture) well in the middle and a waste well. Fluid perfusion is achieved using a combination of the hydrostatic pressure generated by different liquid levels in the wells and the fluid wicking through narrow strips of a cellulose membrane connecting the wells. There is an excellent correspondence between the observed perfusion flow dynamics and the flow simulations based on Darcy's Law. Hepatocytes (C3A cells) cultured for 4 days in the perfusion microplate with no media exchange in the cell culture well had the same viability as hepatocytes exposed to a daily exchange of media. EOC 20 cells that require media conditioned by LADMAC cells were shown to be equally viable in the adjacent cell culture well of the perfusion microplate with LADMAC cells cultured in the source well. Tegafur, a prodrug, when added to primary human hepatocytes in the source well, was metabolized into a cytotoxic metabolite that kills colon cancer cells (HCT 116) cultured in the adjacent cell culture well; no toxicity was observed when only medium was in the source well. These results suggest that the perfusion microplate is a useful tool for a variety of cell culture applications with benefits ranging from labor savings to enabling in vivo-like toxicity studies. PMID:23344077

Goral, Vasiliy N; Zhou, Chunfeng; Lai, Fang; Yuen, Po Ki

2013-03-21

171

Experimental hypersensitivity pneumonitis: transfer with cultured cells.  

PubMed

The importance of antibody and sensitized cells in hypersensitivity pneumonitis (HP) is unknown. In an attempt to create a model suitable for investigation of the mechanisms of HP, we transferred cells and serum from sensitized (Micropolyspora faeni in Freund's adjuvant) strain 2 guinea pigs to naive animals. Cells (peritoneal exudate, lymph node, or spleen) were cultured for 72 hours with either concanavalin A (Con A, 1 microgram/ml) or a soluble extract of M. faeni (10 micrograms/ml). We then injected the cells intravenously (IV) into naive guinea pigs, skin tested with purified protein derivative (PPD), challenged the animals intratracheally (IT) with M. faeni 48 hours after the cell transfer, and killed them 4 days (IT) with M. faeni 48 hours after the cell transfer, and killed them 4 days after IT challenge. We also transferred noncultured cells and antibody-containing serum from sensitized animals. Randomly selected microscopic fields of the lung (150 per animal) were judged to be normal or abnormal. All guinea pigs were maintained in high-efficiency particulate accumulator-filtered air. Compared with control animals that received media IV, there was a substantial increase (P less than 0.01) in the extent of pulmonary abnormalities in the animals receiving lymph node cells or spleen cells cultured with M. faeni, and peritoneal exudate cells cultured with Con A. Findings in recipients of peritoneal exudate cells cultured with M. faeni, or lymph node cells or spleen cells cultured with Con A did not differ from those in the control group. In contrast to cultured cells, noncultured cells and antibody-containing serum did not transfer susceptibility. PPD skin reactivity was present only in recipients of noncultured cells and not in recipients of serum or cultured cells. We conclude that experimental HP can be transferred with cultured cells from sensitized animals and that HP appears to be a cell-mediated process. PMID:3585138

Schuyler, M; Subramanyan, S; Hassan, M O

1987-06-01

172

Culture and characterization of human bone marrow mesenchymal stem cells.  

PubMed

Bone marrow (BM) mesenchymal stem cells (MSCs) are non-hematopoietic cells capable of generating colonies of plastic-adherent marrow mesenchymal cells, each derived from a single cell termed a colony-forming unit fibroblasts (CFU-Fs). In addition to their role in establishing the marrow microenvironment, these cells have been shown to differentiate into several types of mesenchymal and non-mesenchymal lineages. Because of their multipotency, MSCs represent an attractive cellular source in the promising field of cellular therapy. In this chapter, we will focus on culture conditions for human BM MSC expansion and CFU-F assays. We also describe the methodologies to analyze the primary cultures obtained, both at the phenotypic and at the functional levels. Phenotypically, MSCs can be defined with a minimal set of markers as CD31-, CD34-, and CD45-negative cells and CD13-, CD29-, CD73-, CD90-, CD105-, and CD166-positive cells. Functionally, we describe the culture conditions (specific media and cellular preparations) for in vitro differentiation of MSCs into the adipogenic, osteogenic, and chondrogenic lineages. The corresponding colorimetric assays (oil red O, Von Kossa and alizarin red S, and safranin O and alcian blue stains, respectively) are also described. PMID:18085203

Delorme, Bruno; Charbord, Pierre

2007-01-01

173

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

174

Detection of Aneuploidy by a Monochromosomal Hybrid Cell Assay.  

National Technical Information Service (NTIS)

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 c...

S. S. Sandhu R. S. Athwal

1987-01-01

175

Are in vitro estimates of cell diffusivity and cell proliferation rate sensitive to assay geometry?  

PubMed

Cells respond to various biochemical and physical cues during wound-healing and tumour progression. in vitro assays used to study these processes are typically conducted in one particular geometry and it is unclear how the assay geometry affects the capacity of cell populations to spread, or whether the relevant mechanisms, such as cell motility and cell proliferation, are somehow sensitive to the geometry of the assay. In this work we use a circular barrier assay to characterise the spreading of cell populations in two different geometries. Assay 1 describes a tumour-like geometry where a cell population spreads outwards into an open space. Assay 2 describes a wound-like geometry where a cell population spreads inwards to close a void. We use a combination of discrete and continuum mathematical models and automated image processing methods to obtain independent estimates of the effective cell diffusivity, D, and the effective cell proliferation rate, ?. Using our parameterised mathematical model we confirm that our estimates of D and ? accurately predict the time-evolution of the location of the leading edge and the cell density profiles for both assay 1 and assay 2. Our work suggests that the effective cell diffusivity is up to 50% lower for assay 2 compared to assay 1, whereas the effective cell proliferation rate is up to 30% lower for assay 2 compared to assay 1. PMID:24787651

Treloar, Katrina K; Simpson, Matthew J; Sean McElwain, D L; Baker, Ruth E

2014-09-01

176

Culture of Cells from Amphibian Embryos.  

ERIC Educational Resources Information Center

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

Stanisstreet, Martin

1983-01-01

177

Inflight Assay of Red Blood Cell Deformability  

NASA Technical Reports Server (NTRS)

Studies on Soviet and American astronauts have demonstrated that red blood cell production is altered in response to low gravity (g) environment. This is associated with changes in individual red cells including increased mean cell volume and altered membrane deformability. During long orbital missions, there is a tendency for the red cell mass deficit to be at least partly corrected although the cell shape anomalies are not. Data currently available suggest that the observed decrease in red cell mass is the result of sudden suppression of erythropoieses and that the recovery trend observed during long missions reflects re-establishment of erythropoietic homeostasis at a "set point" for the red cell mass that is slightly below the normal level at 1 g.

Ingram, M.; Paglia, D. E.; Eckstein, E. C.; Frazer, R. E.

1985-01-01

178

Human Cell Culture in a Space Bioreactor.  

National Technical Information Service (NTIS)

Microgravity offers new ways of handling fluids, gases, and growing mammalian cells in efficient suspension cultures. In 1976 bioreactor engineers designed a system using a cylindrical reactor vessel in which the cells and medium are slowly mixed. The rea...

D. R. Morrison

1988-01-01

179

Micronucleus assay with tetrad cells of Tradescantia.  

PubMed

The Tradescantia micronucleus assay is being used since almost 50 years for the detection of genotoxins (including carcinogens) in the environment. A large database on the effects of individual compounds and of complex environmental mixtures (soil, air and water) has accumulated. In contrast to other mutagenicity test systems, the effects of low concentrations of heavy metals, radionuclides, certain herbicides and pesticides, and gaseous mutagens can be detected and it is also possible to use the test for in situ biomonitoring studies. The test system has been validated, and standardized protocols have been developed for laboratory experiments and for field studies, which are described in this chapter. PMID:23896890

Mišík, Miroslav; Pichler, Clemens; Rainer, Bernhard; Nersesyan, Armen; Knasmueller, Siegfried

2013-01-01

180

Experimental model for observation of micromotion in cell culture.  

PubMed

It is known that the micromotion between implant and bone inhibits direct bone growth either on or into implant surfaces in vivo. Nevertheless, biocompatibility tests in vitro of biomaterials for bone/implant interfaces are mainly performed under static conditions. This work describes a dynamic, in vitro experimental simulation of the effect of mutual, small-scale implant surface-tissue displacement on adhered cells. Disks of simulated tissue (PVP hydrogel) were subjected to cyclic micromotion ranging from 0 at the center to 1000 microm at the periphery at approximately 13 Hz, relative to biomaterial surfaces or tissue culture polystyrene controls populated with human osteoblasts in standard tissue culture plate wells. The effect of the interfacial micromotion on the number of cells remaining attached was quantitated by XTT assay. The activity level of the remaining cells was determined by an alkaline phosphatase assay, and cell stress was evaluated by nitrogen assay. Significantly more cells (ANOVA) became detached from similarly prepared surfaces of titanium, hydroxyapatite, and alumina compared to the polystyrene control, and detachment from alumina was greater than for the other two materials. The activity of the remaining attached cells was lower as compared to the static (no micromotion) control but not significantly different among the biomaterials. All nitrogen assays were negative, suggesting minimal cell stress occurred. The method is proposed as a useful and discriminating in vitro tool for biocompatibility studies focused on cell adhesion to biomaterials under conditions related to those which exist at the implant/bone interface in vivo, and it allows subsequent studies of the still-viable cells by other methods. PMID:15654711

Lewandowska-Szumie?, Ma?gorzata; Sikorski, Krzysztof; Szummer, Andrzej; Komender, Janusz; Kowalski, Marcin; Daniels, A U

2005-02-15

181

Gametogony of Sarcocystis sp. in Cell Culture  

Microsoft Academic Search

Sexual stages and cystlike bodies of Sarcocystis sp., a protozoan parasite found in muscles of reptiles, birds, and mammals, including man, developed in cell culture. Motile organisms, obtained from leg muscles of wild grackles, were inoculated into cell line cultures of embryonic bovine kidney. Mature micro- and macrogametes and the cystlike forms were found 30 and 42 hours after inoculation,

Ronald Fayer

1972-01-01

182

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...

183

Large-Scale Cell Culture in Biotechnology  

Microsoft Academic Search

The purpose of this article is to review the current state of large-scale cell culture in terms of its applications, problems, and potential. Because of the commercial and proprietary nature of most large-scale cell culture processes, this review does not contain many detailed scientific results although an attempt is made to address some key issues and findings. Much of this

W. R. Arathoon; J. R. Birch

1986-01-01

184

A simple high-content cell cycle assay reveals frequent discrepancies between cell number and ATP and MTS proliferation assays.  

PubMed

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

185

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.

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

2013-01-01

186

Automation of 3-dimensional cell culture in arrayed microfluidic devices  

PubMed Central

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

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

2011-01-01

187

Nanopillar based electrochemical biosensor for monitoring microfluidic based cell culture  

NASA Astrophysics Data System (ADS)

In-vitro assays using cultured cells have been widely performed for studying many aspects of cell biology and cell physiology. These assays also form the basis of cell based sensing. Presently, analysis procedures on cell cultures are done using techniques that are not integrated with the cell culture system. This approach makes continuous and real-time in-vitro measurements difficult. It is well known that the availability of continuous online measurements for extended periods of time will help provide a better understanding and will give better insight into cell physiological events. With this motivation we developed a highly sensitive, selective and stable microfluidic electrochemical glucose biosensor to make continuous glucose measurements in cell culture media. The performance of the microfluidic biosensor was enhanced by adding 3D nanopillars to the electrode surfaces. The microfluidic glucose biosensor consisted of three electrodes---Enzyme electrode, Working electrode, and Counter electrode. All these electrodes were enhanced with nanopillars and were optimized in their respective own ways to obtain an effective and stable biosensing device in cell culture media. For example, the 'Enzyme electrode' was optimized for enzyme immobilization via either a polypyrrole-based or a self-assembled-monolayer-based immobilization method, and the 'Working electrode' was modified with Prussian Blue or electropolymerized Neutral Red to reduce the working potential and also the interference from other interacting electro-active species. The complete microfluidic biosensor was tested for its ability to monitor glucose concentration changes in cell culture media. The significance of this work is multifold. First, the developed device may find applications in continuous and real-time measurements of glucose concentrations in in-vitro cell cultures. Second, the development of a microfluidic biosensor will bring technical know-how toward constructing continuous glucose monitoring devices. Third, the methods used to develop 3D electrodes incorporated with nanopillars can be used for other applications such as neural probes, fuel cells, solar cells etc., and finally, the knowledge obtained from the immobilization of enzymes onto nanostructures sheds some new insight into nanomaterial/biomolecule interactions.

Gangadharan, Rajan

188

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

PubMed

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

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

2012-08-01

189

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

PubMed Central

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

Bernard, Abigail B.; Lin, Chien-Chi

2012-01-01

190

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

191

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

Microsoft Academic Search

We can detect the b-globin gene sickle cell mutation by using an assay based on the ligase chain reaction. The simultaneous amplification of the human growth hor- mone gene in the same reaction serves as a control for the amount of template DNA or amplification effi- ciency. Ligation products, which are biotinylated at one end and tagged with an arbitrary

Antonio A. Reyes; Paola Carrera; Elena Cardillo; Luis Ugozzoli; Jimmie D. Lowery; Ching-I P. Lin; Matthew Go; Maurizio Ferrari; R. Bruce Wallace

192

Using the Drosophila melanogaster D17-c3 cell culture system to study cell motility  

PubMed Central

Cultured Drosophila melanogaster S2 and S2R + cell lines have become important tools for uncovering fundamental aspects of cell biology as well as for gene discovery. Despite their utility, these cell lines are nonmotile and cannot build polarized structures or cell-cell contacts. Here we outline a previously isolated, but uncharacterized, Drosophila cell line named Dm-D17-c3 (or D17). These cells spread and migrate in culture, form cell-cell junctions and are susceptible to RNA interference (RNAi). Using this protocol, we describe how investigators, upon receiving cells from the Bloomington stock center, can culture cells and prepare the necessary reagents to plate and image migrating D17 cells; they can then be used to examine intracellular dynamics or observe loss-of-function RNAi phenotypes using an in vitroscratch or wound healing assay. From first thawing frozen ampules of D17 cells, investigators can expect to begin assaying RNAi phenotypes in D17 cells within roughly 2–3 weeks.

Currie, Joshua D; Rogers, Stephen L

2014-01-01

193

Large-scale cell culture in biotechnology.  

PubMed

The purpose of this article is to review the current state of large-scale cell culture in terms of its applications, problems, and potential. Because of the commercial and proprietary nature of most large-scale cell culture processes, this review does not contain many detailed scientific results although an attempt is made to address some key issues and findings. Much of this summary deals with processes having an established, commercial track record but some attention is given to more recent innovations with interesting potential applications. Reference is made to plant cell culture but the main emphasis is on mammalian cells. PMID:2424083

Arathoon, W R; Birch, J R

1986-06-13

194

Calcium deposition in osteoarthritic meniscus and meniscal cell culture  

PubMed Central

Introduction Calcium crystals exist in the knee joint fluid of up to 65% of osteoarthritis (OA) patients and the presence of these calcium crystals correlates with the radiographic evidence of hyaline cartilaginous degeneration. This study sought to examine calcium deposition in OA meniscus and to investigate OA meniscal cell-mediated calcium deposition. The hypothesis was that OA meniscal cells may play a role in pathological meniscal calcification. Methods Studies were approved by our human subjects Institutional Review Board. Menisci were collected during joint replacement surgeries for OA patients and during limb amputation surgeries for osteosarcoma patients. Calcium deposits in menisci were examined by alizarin red staining. Expression of genes involved in biomineralization in OA meniscal cells was examined by microarray and real-time RT-PCR. Cell-mediated calcium deposition in monolayer culture of meniscal cells was examined using an ATP-induced 45calcium deposition assay. Results Calcium depositions were detected in OA menisci but not in normal menisci. The expression of several genes involved in biomineralization including ENPP1 and ANKH was upregulated in OA meniscal cells. Consistently, ATP-induced calcium deposition in the monolayer culture of OA meniscal cells was much higher than that in the monolayer culture of control meniscal cells. Conclusions Calcium deposition is common in OA menisci. OA meniscal cells calcify more readily than normal meniscal cells. Pathological meniscal calcification, which may alter the biomechanical properties of the knee meniscus, is potentially an important contributory factor to OA.

2010-01-01

195

Hydroxyethyl disulfide as an efficient metabolic assay for cell viability in vitro.  

PubMed

Cell viability assays have a variety of well known practical and technical limitations. All the available approaches have disadvantages, such as non-linearity, high background and cumbersome protocols. Several commonly used tetrazolium chemicals rely upon generation of a colored formazan product formed by mitochondrial reduction of these compounds via phenazine methosulfate (PMS). However, sensitivity is inherently limited because their reduction relies on mitochondrial bioreduction and cellular transport of PMS, as well as accessibility to tetrazolium chemicals. In this study, we identify hydroxethyldisulfide (HEDS) as an inexpensive probe that can measure cellular metabolic activity without the need of PMS. In tissue culture medium, HEDS accurately quantitated metabolically active live cells in a linear manner superior to tetrazolium based and other assays. Cell toxicity produced by chemotherapeutics (cisplatin, etoposide), oxidants (hydrogen peroxide, acetaminophen), toxins (phenyl arsine oxide, arsenite) or ionizing radiation was rapidly determined by the HEDS assay. We found that HEDS was superior to other commonly used assays for cell viability determinations in its solubility, membrane permeability, and intracellular conversion to a metabolic reporter that is readily transported into the extracellular medium. Our findings establish the use of HEDS in a simple, rapid and low cost assay to accurately quantify viable cells. PMID:22321380

Li, Jie; Zhang, Donglan; Ward, Kathleen M; Prendergast, George C; Ayene, Iraimoudi S

2012-06-01

196

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

197

Glycosylation of Fluorophenols by Plant Cell Cultures  

PubMed Central

Fluoroaromatic compounds are used as agrochemicals and released into environment as pollutants. Glycosylation of 2-, 3-, and 4-fluorophenols using plant cell cultures of Nicotiana tabacum was investigated to elucidate their potential to metabolize these compounds. Cultured N. tabacum cells converted 2-fluorophenol into its ?-glucoside (60%) and ?-gentiobioside (10%). 4-Fluorophenol was also glycosylated to its ?-glucoside (32%) and ?-gentiobioside (6%) by N. tabacum cells. On the other hand, N. tabacum glycosylated 3-fluorophenol to ?-glucoside (17%).

Shimoda, Kei; Kubota, Naoji; Kondo, Yoko; Sato, Daisuke; Hamada, Hiroki

2009-01-01

198

Imaging of Cultured Cells by Mass Spectrometry  

Microsoft Academic Search

\\u000a Establishment of an Imaging mass spectrometry (IMS) experimental procedure for cultured cells is an important issue because\\u000a it provides information on localization of various types of biomolecules inside the cell. At present, how to prepare the samples\\u000a from cultured cells for an IMS has been under study. In this section, we present the preparation method for IMS analysis of\\u000a mouse

Hyun Jeong Yang; Yuki Sugiura; Koji Ikegami; Mitsutoshi Setou

199

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

200

Indirect 125I-labeled protein A assay for monoclonal antibodies to cell surface antigens.  

PubMed

An assay for detection of monoclonal hybridoma antibodies against cell surface antigens is described. Samples of spent medium from the hybridoma cultures are incubated in microtest wells with cells, either as adherent monolayers or in suspension. Antibodies bound to surface antigens are detected by successive incubations with rabbit anti-immunoglobulin serum and 125I-labeled protein A from Staphylococcus aureus, followed by autoradiography of the microtest plate or scintillation counting of the individual wells. Particular advantages of this assay for screening hybridomas are: (1) commerically available reagents are used, (2) antibodies of any species and of any immunoglobulin class or subclass can be detected, and (3) large numbers of samples can be screened rapidly and inexpensively. We have used the assay to select hybridomas producing monoclonal antibodies to surface antigens of human melanomas and mouse sarcomas. PMID:316440

Brown, J P; Tamerius, J D; Hellström, I

1979-01-01

201

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

202

Cell-based multiwell assays for the detection of substrate accumulation and oxidation.  

PubMed

We describe multiwell assays for detecting the accumulation as well as the subsequent oxidation of (14)C-labeled substrates in cultured cells. Accumulation is monitored in real time by an established scintillation proximity assay in which the scintillator is embedded in the plate base primarily detecting cell-associated radiolabel. The substrate oxidation assay is a novel variant of previously described experimental approaches aimed at trapping (14)CO(2) produced by isolated enzymes, organelles, or intact cells. This method uses a standard 96-well tissue culture plate and, on top, an inverted filter plate immersed with NaOH that are clamped into a sandwich sealed with a silicon gasket to obtain gas-tight compartments. (14)CO(2) is captured in the filter and quantified by conventional scintillation. We demonstrate both the accumulation and subsequent oxidation of (14)C-labeled substrates in cultured human myotubes, adipocytes, and hepatocytes. Both methods are adaptable for compound screening; at the same time, these protocols provide easy-to-use and time- saving methods for in vitro studies of cellular fuel handling. PMID:17213484

Wensaas, A J; Rustan, A C; Lövstedt, K; Kull, B; Wikström, S; Drevon, C A; Hallén, S

2007-04-01

203

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

PubMed Central

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.

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

2013-01-01

204

Micro-patterned porous substrates for cell-based assays.  

PubMed

In the search for new therapeutic chemicals, lab-on-a-chip systems have recently emerged as innovative and efficient tools for cell-based assays and high throughput screening. Here, we describe a novel, versatile and simple device for cell-based assays at the bench-top. We created spatial variations of porosity on the surface of a membrane filter by microcontact printing with a biocompatible polymer (PDMS). We called such systems Micro-Printed Membranes (?PM). Active compounds dispensed on the porous areas, where the membrane pores are not clogged by the polymer, can cross the membrane and reach cells growing on the opposite side. Only cells immediately below those porous areas could be stimulated by chemicals. We performed proof-of-principle experiments using Hoechst nuclear staining, calcein-AM cell viability assay and destabilization of the cytoskeleton organisation by cytochalasin B. Resulting fluorescent staining properly matched the drops positioning and no cross-contaminations were observed between adjacent tests. This well-less cell-based screening system is highly flexible by design and it enables multiple compounds to be tested on the same cell tissue. Only low sample volumes in the microlitre range are required. Moreover, chemicals can be delivered sequentially and removed at any time while cells can be monitored in real time. This allows the design of complex, sequential and combinatorial drug assays. ?PMs appear as ideal systems for cell-based assays. We anticipate that this lab-on-chip device will be adapted for both manual and automated high content screening experiments. PMID:22434338

Evenou, Fanny; Di Meglio, Jean-Marc; Ladoux, Benoit; Hersen, Pascal

2012-05-01

205

Microvalve-assisted patterning platform for measuring cellular dynamics based on 3D cell culture.  

PubMed

A microfluidic platform to satisfy both 3D cell culture and cell-based assay is required for credible assay results and improved assay concept in drug discovery. In this article, we demonstrate a microvalve-assisted patterning (MAP) platform to provide a new method for investigating cellular dynamics by generating a linear concentration gradient of a drug as well as to realize 3D cell culture in a microenvironment. The MAP platform was fabricated by multilayer soft lithography and several microvalves made it possible to pattern a cell-matrix (scaffold) and to exchange media solutions without breaking cell-matrix structure in a microchannel. This approach provides not only exact fluids control, bubble removal, and stable solution exchange in a microchannel, but also reliable scaffold fabrication and 3D cell culture. In this study, hepatotoxicity tests with human hepatocellular liver carcinoma cells (HepG2) were also performed in real-time monitoring where cell morphologies exposed to different drug concentrations were observed at a time. Compared to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, the MAP platform could be used to reduce drug amount and assay time for cell-based assays as much as 10 and 3 times, respectively. PMID:18942775

Kim, Minseok S; Lee, Wonhye; Kim, Yu Chang; Park, Je-Kyun

2008-12-01

206

A Radioligand Binding Assay for Antitubulin Activity in Tumor Cells  

Microsoft Academic Search

The benzamide RH-5854 is shown to be highly potent toward tumor cells and to arrest nuclear division by a highly specific covalent binding to the &bgr;-subunit of tubulin in the colchicine binding region. Binding of 3H-RH-5854 to &bgr;-tubulin in HCT-116 colon cancer cells is saturable and has been exploited in the development of a cell-based competitive binding assay, which allows

David H. Young; Fernando M. Rubio; Paul O. Danis

2006-01-01

207

Broad-range real-time PCR assay for the rapid identification of cell-line contaminants and clinically important mollicute species  

Microsoft Academic Search

Polymerase chain reaction assays have become widely used methods of confirming the presence of Mollicutes species in clinical samples and cell cultures. We have developed a broad-range real-time PCR assay using the locked nucleic acid technology to detect mollicute species causing human infection and cell line contamination. Primers and probes specifically for the conserved regions of the mycoplasmal tuf gene

Melanie Störmer; Tanja Vollmer; Birgit Henrich; Knut Kleesiek; Jens Dreier

2009-01-01

208

Miniaturisation and validation of a cell-based assay for screening of Ca2+ channel modulators.  

PubMed

Voltage-operated calcium channels (VOCCs) play a significant role in the regulation of intracellular calcium concentrations in cardiovascular, neuronal and skeletal tissues. Therefore, physiologically relevant screening methods for calcium channel modulators are required. A 45Ca2+ uptake assay based on clonal rat pituitary cell line GH4C1, possessing L-type VOCCs, was miniaturised into a 96-well plate format. The assay was validated by known Ca2+ channel blockers, verapamil and nimodipine (IC50 values 3.4 and 0.007 microM, respectively) and by a set of natural compounds and their synthetic derivatives. The results were consistent with our previous data and demonstrated the reliability of the assay. The signal-to-background ratio was 3.9 +/- 0.4, signal-to-noise ratio 10.3 +/- 2.3, Z' factor 0.59 +/- 0.10, and day-to-day variability in positive control values 5%. Furthermore, experiments were also made on a Biomek FX workstation to evaluate the suitability of the assay for automation. With minor modifications the assay is applicable, e.g. for studying possible Ca2+ channel activators in detail. The established 96-well plate assay modification for screening of calcium channel modulators reduces considerably the time, labour and resources needed for cell culture and experiments, and has significant advantages in terms of automation suitability and overall cost-efficiency. PMID:15165754

Tammela, Päivi; Vuorela, Pia

2004-06-30

209

Cell-based assay protocol for the prognostic prediction of idiopathic scoliosis using cellular dielectric spectroscopy.  

PubMed

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells' responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days. PMID:24192997

Akoume, Marie-Yvonne; Franco, Anita; Moreau, Alain

2013-01-01

210

Methods for Maintaining Insect Cell Cultures  

PubMed Central

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

Lynn, Dwight E.

2002-01-01

211

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

Microsoft Academic Search

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

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

1966-01-01

212

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

213

Cell Culture Derived AgMNPV Bioinsecticide: Biological Constraints and Bioprocess Issues  

Microsoft Academic Search

We have studied parameters for optimizing the Spodoptera frugiperda (Sf9) cell culture and viral infection for the production of Anticarsia gemmatalis multiple nucleopolyhedrosis virus (AgMNPV) polyhedra inclusion bodies (PIBs) in shaker-Schott or spinner bottles and bioreactors.\\u000a We have assayed the kLa of the systems, initial cell seeding, cell culture volume, dissolved oxygen (DO), multiplicity of infection (MOI), nutrients\\u000a consumption, and

Valeria M. Rodas; Fabiano H. Marques; Marcelo T. Honda; Daniela M. Soares; Soraia A. C. Jorge; Marta M. Antoniazzi; Claudia Medugno; Maria E. B. Castro; Bergmann M. Ribeiro; Marlinda L. Souza; Aldo Tonso; Carlos A. Pereira

2005-01-01

214

In vitro osteogenesis assays: influence of the primary cell source on alkaline phosphatase activity and mineralization.  

PubMed

In trabecular bone fracture repair in vivo, osteogenesis occurs through endochondral ossification under hypoxic conditions, or through woven bone deposition in the vicinity of blood vessels. In vitro osteogenesis assays are routinely used to test osteoblastic responses to drugs, hormones, and biomaterials for bone and cartilage repair applications. These cell culture models recapitulate events that occur in woven bone synthesis, and are carried out using primary osteoblasts, osteoblast precursors such as bone marrow-derived mesenchymal stromal cells (BMSCs), or various osteoblast cell lines. With time in culture, cell differentiation is typically assessed by examining levels of alkaline phosphatase activity (an early osteoblast marker) and by evaluating the assembly of a collagen (type I)-containing fibrillar extracellular matrix that mineralizes. In this review, we have made a comparative analysis of published osteogenic assays using calvarial cells, calvaria-derived cell lines, and bone marrow stromal cells. In all of these cell types, alkaline phosphatase activity shows similar progression over time using a variety of osteogenic and mineralizing media conditions; however, levels of alkaline phosphatase activity are not proportional to observed mineralization levels. PMID:18842361

Hoemann, C D; El-Gabalawy, H; McKee, M D

2009-06-01

215

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.

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

2013-01-01

216

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

NASA Astrophysics Data System (ADS)

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

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

2009-05-01

217

Progress in Cell Based Assays for Botulinum Neurotoxin Detection  

PubMed Central

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.

2013-01-01

218

A user-friendly, highly sensitive assay to detect the IFN-? secretion by T cells  

PubMed Central

Objectives Development of a user-friendly test alternative to ELISA-based assays to detect IFN-? by in vitro cultured peripheral blood mononuclear cells (PBMC) stimulated with pathogen-derived antigens. Design and methods The molecular components of an operational IFN-? ELISA-based test were applied in a lateral flow (LF) immuno-sandwich assay using up-converting phosphor (UCP) reporter particles. The analytical sensitivity of the UCP-LF IFN-? assay (ULIGA) was determined and the assay was qualitatively validated with a selection of 60 supernatants derived from PBMC cultures stimulated with M. leprae derived antigens, mitogen or medium alone. Results ULIGA indicated an analytical sensitivity better than 2 pg/mL, and demonstrated four orders of magnitude dynamic range. The assay correlated well with the IFN-? ELISA. Conclusions ULIGA allows detection well below the cutoff value (100 pg/mL) used to define positive responses in the IFN-? ELISA. The test procedure is less demanding in respect to equipment and labor, and is suited for testing single samples.

Corstjens, Paul L.A.M.; Zuiderwijk, Michel; Tanke, Hans J.; van der Ploeg-Schip, Jolien; Ottenhoff, Tom H.M.; Geluk, Annemiek

2008-01-01

219

Oscillatory behavior of cells in tissue culture.  

NASA Astrophysics Data System (ADS)

Fibroblasts and epithelial cells organize themselves in distinct patterns in tissue culture which indicates that neighboring cells communicate. A striking example of such communication is the oscillatory behavior of Madin-Darby canine kidney (MDCK) cells reported here. These oscillations were discovered using a biosensor referred to as ECIS (Electric Cell-substrate Impedance Sensing). In this measurement cells are seeded out on a small electrode deposited at the bottom of a tissue culture well and immersed in ordinary culture medium. By measuring the changes in the impedance of the electrode as a function of time, many important properties of the cells on the electrode can be inferred, such as motion, morphology changes and membrane capacitance. The impedance oscillations of MDCK cells were observed with highly confluent cell layers, where the approximately 100 cells on the electrode acted in unison. The communication between cells can be demonstrated directly by a variation of the ECIS concept, where cells are cultured on two closely spaced electrodes. The impedance fluctuations are measured independently on each electrode and compared by using a cross-correlation function.

Giaever, Ivar; Linton, Michael F. A.; Keese, Charles R.

1998-03-01

220

In vitro assay of bacterial adhesion onto mammalian epithelial cells.  

PubMed

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells(1). These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'(2). Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories(3,4). These assays are now practiced by most laboratories working on bacterial pathogenesis. Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 2787(5), a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787?aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before(6). PMID:21633326

Letourneau, Jason; Levesque, Cynthia; Berthiaume, Frederic; Jacques, Mario; Mourez, Michael

2011-01-01

221

High-content adhesion assay to address limited cell samples†  

PubMed Central

Cell adhesion is a broad topic in cell biology that involves physical interactions between cells and other cells or the surrounding extracellular matrix, and is implicated in major research areas including cancer, development, tissue engineering, and regenerative medicine. While current methods have contributed significantly to our understanding of cell adhesion, these methods are unsuitable for tackling many biological questions requiring intermediate numbers of cells (102–105), including small animal biopsies, clinical samples, and rare cell isolates. To overcome this fundamental limitation, we developed a new assay to quantify the adhesion of ~102–103 cells at a time on engineered substrates, and examined the adhesion strength and population heterogeneity via distribution-based modeling. We validated the platform by testing adhesion strength of cancer cells from three different cancer types (breast, prostate, and multiple myeloma) on both IL-1? activated and non-activated endothelial monolayers, and observed significantly increased adhesion for each cancer cell type upon endothelial activation, while identifying and quantifying distinct subpopulations of cell-substrate interactions. We then applied the assay to characterize adhesion of primary bone marrow stromal cells to different cardiac fibroblast-derived matrix substrates to demonstrate the ability to study limited cell populations in the context of cardiac cell-based therapies. Overall, these results demonstrate the sensitivity and robustness of the assay as well as its ability to enable extraction of high content, functional data from limited and potentially rare primary samples. We anticipate this method will enable a new class of biological studies with potential impact in basic and translational research.

Warrick, Jay W.; Young, Edmond W. K.; Schmuck, Eric G.; Saupe, Kurt W.

2013-01-01

222

Modeling of cell cultures in perfusion bioreactors.  

PubMed

Cultivating cells and tissues in bioreactors is a critical step in forming artificial tissues or organs prior to transplantation. Among various bioreactors, the perfusion bioreactor is known for its enhanced convection through the cell-scaffold constructs. Knowledge of mass transfer is essential for controlling the cell culture process; however, obtaining this information remains a challenging task. In this research, a novel mathematical model is developed to represent the nutrient transport and cell growth in a 3-D scaffold cultivated in a perfusion bioreactor. Numerical methods are employed to solve the equations involved, with a focus on identifying the effect of factors such as porosity, culturing time, and flow rate, which are controllable in the scaffold fabrication and culturing process, on cell cultures. To validate the new model, the results from the model simulations were compared to the experimental results extracted from the literature. With the validated model, further simulations were carried out to investigate the glucose and oxygen distribution and the cell growth within the cell-scaffold construct in a perfusion bioreactor, thus providing insight into the cell culture process. PMID:22772976

Yan, X; Bergstrom, D J; Chen, X B

2012-09-01

223

Phenotypic change of human cultured meningioma cells.  

PubMed

One objection to using cell cultures for studying the proliferation of tumors is the potential for phenotypic changes that may occur in vitro. Here, we compared the antigen pattern expression of cultured meningioma cells with that of the primary tumor. Cell cultures established from 9 intracranial meningiomas and deparaffinized sections of the resected tumors were analyzed for immunophenotyping with the following antibodies: vimentin, cytokeratin, epithelial membrane antigen, S-100, neuron-specific enolase, synaptophisin, factor VIII-related antigen, CD4, CD31, CD34, CD45RB, CD68-PGM1, CD68-KP, and myeloid/histiocyte antigen (MAC387). Overall, the cultured meningioma cells retained the main feature of the primary tumor, being positive both for mesenchymal antigens and for epithelial antigens. Interestingly, the cultured meningioma cells abundantly expressed the CD68 antigens at early passage. The CD68 antigens, which are normally found on hematopoietic cells like macrophages and monocytes, were not detectable on meningioma cells in situ. Our results show that phenotypic changes on human meningioma cells may occur in vitro. This phenomenon suggests caution when transposing the in vitro results to the in vivo condition. PMID:11131990

Pallini, R; Casalbore, P; Mercanti, D; Maggiano, N; Larocca, L M

2000-08-01

224

Reactivity of alveolar epithelial cells in primary culture with type I cell monoclonal antibodies.  

PubMed

An understanding of the process of alveolar epithelial cell growth and differentiation requires the ability to trace and analyze the phenotypic transitions that the cells undergo. This analysis demands specific phenotypic probes to type II and, especially, type I pneumocytes. To this end, monoclonal antibodies have been generated to type I alveolar epithelial cells using an approach designed to enhance production of lung-specific clones from a crude lung membrane preparation. The monoclonal antibodies were screened by a combination of enzyme-linked immunosorbent assay and immunohistochemical techniques, with the determination of type I cell specificity resting primarily on immunoelectron microscopic localization. Two of these new markers of the type I pneumocyte phenotype (II F1 and VIII B2) were used to analyze primary cultures of type II cells growing on standard tissue culture plastic and on a variety of substrata reported to affect the morphology of these cells in culture. On tissue culture plastic, the antibodies fail to react with early (days 1 to 3) type II cell cultures. The cells become progressively more reactive with time in culture to a plateau of approximately 6 times background by day 8, with a maximum rate of increase between days 3 and 5. This finding is consistent with the hypothesis that type II cells in primary culture undergo at least partial differentiation into type I cells. Type II cells grown on laminin, which reportedly delays the loss of type II cell appearance, and on fibronectin, which has been reported to facilitate cell spreading and loss of type II cell features, develop the type I cell markers during cultivation in vitro with kinetics similar to those on uncoated tissue culture plastic. Cells on type I collagen and on tissue culture-treated Nuclepore filters, which have been reported to support monolayers with type I cell-like morphology, also increase their expression of the II F1 and VIII B2 epitopes around days 3 to 5. Taken together with available morphologic information, these data suggest that expression of different alveolar epithelial cell phenotypic markers by type II cells in primary culture may be independently regulated. The monoclonal antibody probes described in this report should prove useful in the continued investigation of the mechanisms and regulation of alveolar epithelial cell differentiation. PMID:1540393

Danto, S I; Zabski, S M; Crandall, E D

1992-03-01

225

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

226

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

PubMed

Pyrogen induced secretion of interleukin 6 (IL-6) in Mono Mac 6 (MM6) cells was measured. The ability of the MM6 cell culture to detect pyrogens was compared to the Limulus amoebocyte lysate (LAL) test and isolated mononuclear cells (MNC). The detection limit of MM6 for lipopolysaccharide (LPS) and Staphylococcus aureus was comparable to that of MNC. Aspergillus niger and Candida albicans induced IL-6 in isolated MNC, but not in MM6. The detection limit for Salmonella typhimurium in the MM6 assay was comparable to that of the LAL assay. As expected, S. aureus and C. albicans did not show any LAL activity. A. niger and Influenza virus showed some activity in the LAL test, but could not be detected by MM6 cells. In conclusion, the MM6 assay is a good supplement to the current pyrogen assays for detection of LPS, S. aureus and S. typhimurium, but the MM6 assay could not detect A. niger, C. albicans and Influenza virus. PMID:10564840

Moesby, L; Jensen, S; Hansen, E W; Christensen, J D

1999-11-30

227

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

PubMed

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

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

2014-04-15

228

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

EPA Science Inventory

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

229

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, ...

230

Biomimetic macroporous hydrogel scaffolds in a high-throughput screening format for cell-based assays.  

PubMed

Macroporous hydrogels (MHs) hold great promise as scaffolds in tissue engineering and cell-based assays. In this study, the possibility of combination of three-dimensional (3D) cell culture with a miniaturized screening format was demonstrated on human colon cancer HCT116, human acute myeloid leukemia KG-1 cells, and embryonic fibroblasts cultured on MHs (12.5 mm x 7.1 mm I.D.) in a 96-minicolumn plate format. MHs were prepared by cryogelation technique and functionalized by coating with type I collagen and by copolymerization with agmatine-based mimetic of cell adhesive peptide RGD (abRGDm). Cancer cells formed multicellular aggregates while fibroblasts formed adhesions on abRGDm-containing and collagen-MHs but not on plain MHs, as was demonstrated by scanning electron microscopy. HCT116 and KG-1 cells grown as aggregates were more resistant to the treatment with cis-diaminedichloroplatinum (II) (cisplatin) and cytosine 1-beta-D-arabinofuranoside (Ara-C), respectively, during the first 18-24 h of incubation, than single cells grown on unmodified MH. HCT116 cells grown as 2D cultures in conventional 96-well tissue culture plates were 1.5- to 3.5-fold more sensitive to the treatment with 70 microM cisplatin than cells in 3D cultures in functionalized MHs. Further development of the described experimental system including matching of a specific cell type with appropriate extracellular matrix (ECM) components and 3D cocultures on ECM-modified MHs may provide a realistic in vitro experimental model for high-throughput toxicity tests. PMID:19194952

Dainiak, Maria B; Savina, Irina N; Musolino, Isabella; Kumar, Ashok; Mattiasson, Bo; Galaev, Igor Yu

2008-01-01

231

Early Detection of Negative BACTEC MGIT 960 Cultures by PCR-Reverse Cross-Blot Hybridization Assay  

PubMed Central

We evaluated the efficacy of a PCR-reverse cross-blot hybridization assay, a test which permits identification of mycobacteria by means of species-specific probes and a Mycobacterium-specific probe, for early detection of negative BACTEC MGIT 960 mycobacterial cultures. Aliquots of 549 cultures were collected 7 days after the culture media were inoculated with various clinical specimens and tested with the molecular assay. PCR results were compared to those obtained at the end times with the BACTEC MGIT 960 system. Of the 549 specimens analyzed, 484 were found to be negative and 64 were found positive by both methods; one specimen, found to be positive by the BACTEC MGIT 960 system, was identified as negative by the molecular assay. In view of its high negative predictive value (99.8%), the PCR-reverse cross-blot hybridization assay appears to be a valid tool for early detection of negative BACTEC MGIT 960 cultures.

Romano, Lucio; Sanguinetti, Maurizio; Posteraro, Brunella; Ardito, Fausta; Gesu, Giovanni; Schito, Anna Maria; Fadda, Giovanni

2002-01-01

232

A sensible technique to detect mollicutes impurities in human cells cultured in GMP condition.  

PubMed

In therapeutic trials the use of manipulated cell cultures for clinical applications is often required. Mollicutes microorganism contamination of tissue cultures is a major problem because it can determine various and severe alterations in cellular function. Thus methods able to detect and trace cell cultures with Mollicutes contamination are needed in the monitoring of cells grown under good manufacturing practice conditions, and cell lines in continuous culture must be tested at regular intervals. We here describe a multiplex quantitative polymerase chain reaction assay able to detect contaminant Mollicutes species in a single-tube reaction through analysis of 16S-23S rRNA intergenic spacer regions and Tuf and P1 cytoadhesin genes. The method shows a sensitivity, specificity, and robustness comparable with the culture and the indicator cell culture as required by the European Pharmacopoeia guidelines and was validated following International Conference on Harmonization guidelines and Food and Drug Administration requirements. PMID:24740225

Ugolotti, Elisabetta; Vanni, Irene

2014-01-01

233

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.

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

2011-01-01

234

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.

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

1996-01-01

235

Quantitatve assay of dissociated tissue cell motility in vitro  

Microsoft Academic Search

Summary  A simple method for quantitating the motile properties of a dissociated tissue cell preparation is presented. A cell population\\u000a is allowed to grow over a glass cover slip coated with Scarlet Red-containing Formvar. At confluence, the preparation is cut\\u000a with a razor to remove a portion of the cells and the underlying pigmented Formvar and then returned to culture conditions.

K. S. Stenn

1980-01-01

236

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

PubMed

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

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

2010-08-01

237

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

PubMed

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

Liu, Tianqing; Winter, Marnie; Thierry, Benjamin

2014-07-01

238

Hepatitis E virus cell culture models.  

PubMed

Early studies reported the propagation of hepatitis E virus (HEV) in either primary hepatocytes or several established cell lines, but replication was inefficient. Efficient cell culture systems for HEV in PLC/PRF/5 and A549 cells have recently been established, using inoculum of fecal suspensions with high HEV loads, originally obtained from patients with genotype 3 HEV (the JE03-1760F strain, 2.0×10(7) copies/ml) or genotype 4 HEV (the HE-JF5/15F strain, 1.3×10(7) copies/ml), and many generations were successfully propagated in serial passages of culture supernatant. In addition, a full-length infectious cDNA clone (pJE03-1760F/wt) of the JE03-1760F strain was constructed, which can replicate efficiently in PLC/PRF/5 and A549 cells. A derivative ORF3-deficient mutant revealed that the ORF3 protein of HEV is responsible for virion egress from infected cells and is present on the surface of released HEV particles, which is associated with lipids. Various HEV strains with high loads of ?10(5) copies/ml in circulating blood were also propagated efficiently in PLC/PRF/5 and A549 cells. This paper reviews the road map toward the development of efficient cell culture systems for a wide variety of HEV strains and introduces the current knowledge on virion egress obtained by cell culture models. PMID:21316402

Okamoto, Hiroaki

2011-10-01

239

Interaction of nanobacteria with cultured mammalian cells  

Microsoft Academic Search

Nanobacteria were recently isolated from human blood and commercial fetal bovine serum (FBS) and were located in the ?-2 subgroup of proteobacteria based upon their 16S rRNA gene sequence. They can be cultured even in the absence of mammalian cells, and have extraordinary properties, like very slow growth rate and an impermeable cell wall, making their detection difficult by standard

Neva Çiftçioglu; E. Olavi Kajander

1998-01-01

240

Transferring isolated mitochondria into tissue culture cells.  

PubMed

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

Yang, Yi-Wei; Koob, Michael D

2012-10-01

241

Evaluation of the Duopath Legionella Lateral Flow Assay for Identification of Legionella pneumophila and Legionella Species Culture Isolates  

Microsoft Academic Search

Duopath Legionella (Merck KGaA, Darmstadt, Germany) is a new immunochromatographic assay for the simultaneous identification of cultured L. pneumophila and Legionella species other than L. pneumophila .I n tests of 89 L. pneumophila strains and 87 Legionella strains other than L. pneumophila representing 41 different species, Duopath and a widely used latex agglutination assay detected L. pneumophila with 100% and

Jurgen Herbert Helbig; Paul Christian Luck; Britta Kunz; Andreas Bubert

2006-01-01

242

Integration of cell culture and microfabrication technology.  

PubMed

Recent progress in cell culture and microfabrication technologies has contributed to the development of cell-based biosensors for the functional characterization and detection of drugs, pathogens, toxicants, and odorants. The cell-based biosensors are composed of two transducers, where the primary transducer is cellular and the secondary transducer is typically electrical. Advances in gene manipulation and cell culture techniques have contributed to the development of the cell as a transducer, while microfabrication techniques have been applied to the development of integrating the cell with the second transducer. Cellular patterning using microfabrication techniques is essential for cell-based biosensors, cell culture analogues, tissue engineering, and fundamental studies of cell biology. The photolithographic technique is highly developed and has been widely used for patterning cells. Recently, a set of alternative techniques, largely based on soft lithoghraphy, has been developed for biological applications. Those techniques include microcontact printing, microfluidic patterning using microchannels, and laminar flow patterning. A classical metallic stencil patterning method has been improved by employing a rubber-like stencil. These cellular micropatterning techniques have been usefully employed to understand questions in fundamental cell biology, especially cellular interactions with various materials and other cells. Using these micropatterning tecchniques and insights into the interaction of cellular biology with surfaces, a wide array of biosensors have been developed. In this manuscript examples of cell-based biosensors are described. Neurons have a great potential for use in a cell-based biosensor because they are electrically excitable cells, from which electrical signals are generated with the binding of detecting molecules. Consequently, the electrical signals generated in the cell can be determined in a noninvasive manner. A microphysiometer is a device to detect functional responses from cells by measuring the change of extracellular pH. The main application of the microphysiometer is the analysis of functional responses of cells upon receptor stimulation. Development of a microscale cell culture analogue system, an in vitro animal or human surrogate, is another promising area using cell culture and microfabrication technologies. Such devices are potentially very useful in the fields of toxicology and drug testing because they may increase the accuracy of in vitro predictions, simplify testing procedures, and reduce the cost of such tests, allowing many more tests to be done with a limited set of resources. PMID:12675556

Park, Tai Hyun; Shuler, Michael L

2003-01-01

243

Limiting-dilution transplantation assays in mammary stem cell studies.  

PubMed

Mammary reconstitution assays can be used to measure the stem cell frequency within an epithelial population by transplanting increasingly diluted single-cell preparations of the population of interest. There are fundamental steps in the single-cell isolation protocol which are directly related to the number of single epithelial cells obtained. Once single-cell suspensions have been obtained, serial dilutions are prepared and transplanted into the cleared fat pads of the host mice. After 8-10 weeks, the transplanted fat pads are reevaluated for the presence of epithelial outgrowths. Based on the frequency of no outgrowth for each one of the transplanted dilutions, it is possible to estimate the frequency of mammary repopulating cells present in a given cell population. Here, we give details on how to carry out all these steps. PMID:20405357

Illa-Bochaca, Irineu; Fernandez-Gonzalez, Rodrigo; Shelton, Dawne N; Welm, Bryan E; Ortiz-de-Solorzano, Carlos; Barcellos-Hoff, Mary Helen

2010-01-01

244

Classical human epidermal keratinocyte cell culture.  

PubMed

It has been more than 30 years since the serial cultivation of human keratinocytes in monolayer culture was first described by Rheinwald and Green. Initially, isolation of primary keratinocytes from disaggregated human skin tissue and subsequent propagation was promoted through use of replication-inactivated murine fibroblast feeder layers. Since then numerous advances have been made to the cultivation of human keratinocytes in both two-dimensional monolayer and three-dimensional organotypic culture. Monolayer culture facilitates keratinocyte proliferation, whereas organotypic culturing techniques promote keratinocyte differentiation using conditions permissive for stratification. The protocols presented here describe traditional culturing methods, providing guidance for isolation and serial cultivation of primary human keratinocytes and dermal fibroblasts, as well as the use of these cells types for generation of stratified skin tissue. PMID:23097107

Rasmussen, Cathy; Thomas-Virnig, Christina; Allen-Hoffmann, B Lynn

2013-01-01

245

Cell-based reporter gene assay for therapy-induced neutralizing antibodies to interferon-beta in multiple sclerosis.  

PubMed

Patients with therapy-induced neutralizing antibodies (NAbs) to interferon-beta (IFN-?) have reduced responses to IFN-? treatment, resulting in higher relapse rates, increased magnetic resonance imaging activity, and a higher risk of disease progression. A functional assay was employed for both screening and titering of IFN-? NAbs utilizing a human cell line transfected with a luciferase reporter gene responsive to IFN-?. This assay demonstrated 100% sensitivity and specificity compared with the traditional cytopathic effect (CPE) assay and normal donor specimens. Additionally, 183 patients with multiple sclerosis (MS) undergoing therapy with IFN-? were tested in the reporter gene assay. Percent positivity for NAbs to the IFN-? was as follows: Avonex (1?) 26.5%, Rebif (1?) 34.1%, and Betaseron (1?) 31.8%. The IFN-? reporter gene assay showed excellent correlation with the well-established CPE assay offering clear advantages. The 50% false-positivity rate typically seen in enzyme-linked immunosorbent assays could be eliminated by using a functional assay for both screening and titering. Results can be reported within 20 h, and the cell line is cryopreserved, eliminating the need to maintain live viral and cell cultures. The use of this functional assay should be a valuable tool for detecting and monitoring the presence of NAbs in IFN-?-treated patients with MS. PMID:23153300

Martins, Thomas B; Rose, John W; Gardiner, Gareth L; Kusukawa, Noriko; Husebye, Dee; Hill, Harry R

2013-02-01

246

Bovine chromaffin cell cultures as model to study organophosporus neurotoxicity.  

PubMed

Based on the high level of phenyl valerate esterase activities, and in particular of neuropathy target esterase (NTE) found in bovine adrenal medulla, chromaffin cells culture have been proposed as an alternative model for the study of organophosphorus neurotoxicity. Organophosphorus-induced polyneuropathy is a syndrome related to the inhibition and further modification by organophosphorus compounds of NTE (a protein that displays phenyl valerate esterase activity resistant to mipafox and sensitive to paraoxon). Total phenyl valerate esterase activities found in homogenate, particulate and soluble fractions of bovine adrenal medulla were 5200+/-35, 5000+/-280 and 1700+/-260 mU/g tissue, respectively. Cultured chromaffin cells displayed a total hydrolysing activity of 41+/-5 mU/10(6) cells. Homogenates of bovine adrenal medulla displayed only about 6% of activity sensitive to paraoxon. Most of the phenyl valerate esterase activity inhibited by mipafox (a neuropathy inducing compound) was found in particulate fraction. Cultured chromaffin cells displayed kinetics of inhibition by mipafox similar to the kinetics displayed by homogenates of bovine adrenal medulla. We conclude that NTE could be assayed in this system by only using one inhibitor (mipafox) instead of two (paraoxon and mipafox). Also, the proposal is supported of using chromaffin cells as in vitro model for the study of the role of NTE and related esterases in organophosphorus-induced polyneuropathy. PMID:15177651

Quesada, E; Sogorb, M A; Vilanova, E; Carrera, V

2004-06-15

247

Hydrogels as extracellular matrix mimics for 3D cell culture  

Microsoft Academic Search

Methods for culturing mammalian cells ex vivo are increasingly needed to study cell and tissue physiology and to grow replacement tissue for regenerative medicine. Two-dimensional culture has been the paradigm for typical in vitro cell culture; however, it has been demonstrated that cells behave more natively when cultured in three- dimensional environments. Permissive, synthetic hydrogels and promoting, natural hydrogels have

Mark W. Tibbitt; Kristi S. Anseth

2009-01-01

248

Detection of maternal cell contamination in amniotic fluid cell cultures using fluorescent labelled microsatellites.  

PubMed

A rapid PCR based assay was used to ascertain the presence of maternal cell contamination (MCC) in amniotic fluid cell cultures and to exclude MCC in cases where cytogenetic analysis was possible only from one primary cell culture. Six 6-carboxyfluorescein (FAM) and three 6-carboxyfluorescein hexachloride (HEX) labelled primer sets were used to amplify two tetra- and seven dinucleotide repeat polymorphisms. The PCR amplifications were multiplexed in (three) three primer set reactions and visualised on an Applied Biosystems 373A sequencer running Genescan 672 software. The microsatellite products obtained from 200 amniotic fluid cell cultures where the karyotype was female were compared against corresponding maternal blood PCR products. A single case of MCC was detected indicating the usefulness of such assays. We suggest that screening for MCC should be considered in instances where the amniotic fluid sample is bloodstained or was obtained with difficulty, or where the karyotype is female and chromosome analysis is not possible from more than one primary cell culture. PMID:7897630

Smith, G W; Graham, C A; Nevin, J; Nevin, N C

1995-01-01

249

Effects of ochratoxin A on DNA evaluated in vitro with the single cell gel electrophoresis (Comet assay)  

Microsoft Academic Search

In cell cultures of Madin Darby canine kidney (MDCK) cells, the mycotoxin ochratoxin A (OTA) induced single strand breaks\\u000a (ssb) in a concentration dependent manner detected with the single cell gel electrophoresis (Comet assay). When an external\\u000a metabolizing enzyme system (S9-mix from rat liver) was added, this genotoxic effect was significantly stronger. By addition\\u000a of methotrexate (MT), a substrate of

S Lebrun; W Föllmann

2001-01-01

250

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

251

Shock wave application to cell cultures.  

PubMed

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to target cells or tissue without any subsequent damage. Therefore, in vitro experiments are of increasing interest. Various methods of applying shock waves onto cell cultures have been described. In general, all existing models focus on how to best apply shock waves onto cells. However, this question remains: What happens to the waves after passing the cell culture? The difference of the acoustic impedance of the cell culture medium and the ambient air is that high, that more than 99% of shock waves get reflected! We therefore developed a model that mainly consists of a Plexiglas built container that allows the waves to propagate in water after passing the cell culture. This avoids cavitation effects as well as reflection of the waves that would otherwise disturb upcoming ones. With this model we are able to mimic in vivo conditions and thereby gain more and more knowledge about how the physical stimulus of shock waves gets translated into a biological cell signal ("mechanotransduction"). PMID:24747842

Holfeld, Johannes; Tepeköylü, Can; Kozaryn, Radoslaw; Mathes, Wolfgang; Grimm, Michael; Paulus, Patrick

2014-01-01

252

The consensus mechanics of cultured mammalian cells  

PubMed Central

Although understanding cells' responses to mechanical stimuli is seen as increasingly important for understanding cell biology, how to best measure, interpret, and model cells' mechanical properties remains unclear. We determine the frequency-dependent shear modulus of cultured mammalian cells by using four different methods, both unique and well established. This approach clarifies the effects of cytoskeletal heterogeneity, ATP-dependent processes, and cell regional variations on the interpretation of such measurements. Our results clearly indicate two qualitatively similar, but distinct, mechanical responses, corresponding to the cortical and intracellular networks, each having an unusual, weak power-law form at low frequency. The two frequency-dependent responses we observe are remarkably similar to those reported for a variety of cultured mammalian cells measured with different techniques, suggesting it is a useful consensus description. Finally, we discuss possible physical explanations for the observed mechanical response.

Hoffman, Brenton D.; Massiera, Gladys; Van Citters, Kathleen M.; Crocker, John C.

2006-01-01

253

Media for culture of mammalian cells.  

PubMed

When mammalian cells are cultured in vitro, the investigator is attempting to reproduce the physiological environment in order to maintain and analyze normal functions and responses. The culture medium is an essential component of the in vitro environment and must be selected or designed with care. This unit provides guidelines for design of serum-containing and serum-free media, selective and specialty media, and media for growth under special conditions such as soft-agar growth. PMID:18228290

Sato, J D; Kan, M

2001-05-01

254

Canine coronavirus induces apoptosis in cultured cells  

Microsoft Academic Search

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

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

2007-01-01

255

Effects of abscisic acid on the secondary metabolism of cultured Onosma paniculatum cells  

Microsoft Academic Search

ABA addition to B5 or M9 medium at the concentrations from 0.1 to 5.0 mg\\/l suppressed growth of Onosma paniculatum cells. The addition of these ABA concentrations to M9 medium also significantly decreased the formation of shikonin and its\\u000a derivatives in the cultured cells during the entire course of culturing. The enzyme activity assay showed that, on the 4th\\u000a day

D.-Y. Sun; Z.-J. Yin; Sh.-J. Wu; J. Su; Sh. Shi; H. Wu; F.-H. Xiao; J.-L. Qi; Zh. Liu; Y.-J. Pang; H.-G. Shen; Y.-H. Yang

2007-01-01

256

Detection of nonhemagglutinating influenza a(h3) viruses by enzyme-linked immunosorbent assay in quantitative influenza virus culture.  

PubMed

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

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

2014-05-01

257

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

PubMed Central

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

2010-01-01

258

Evaluation of the enterococci indicator in biosolids using culture-based and quantitative PCR assays.  

PubMed

The utility of the enterococci indicator for measuring biosolids quality was evaluated in biosolids from 22 U.S. wastewater treatment facilities. Enterococci were enumerated using 23S rRNA quantitative PCR (qPCR) and membrane filtration with mEI-agar culture analyses in biosolids collected after mesophilic anaerobic digestion (MAD, class B, 13 treatment plants), composting (class A, 10 treatment plants), and temperature-phased anaerobic digestion (TPAD, class A, six treatment plants). Enterococci qPCR and culture values were not significantly different for a given treatment (P>0.05, paired t-test) and both assays showed differences in biosolid treatment effectiveness-anaerobic digestion treatments averaged 5-5.5log genomic units (GU) and colony forming units (CFU)/dry g while composting decreased enterococci on average to 3.7logGU and 3.8logCFU/dry g. Only in class A TPAD biosolids dewatered with a belt-filter press were culture values significantly lower than qPCR values (1.7logCFU/dryg vs. 5GU/dryg). Further investigation of compost inactivation was compared for enterococci and other fecal indicators (n=5 treatment plants)-the enterococci indicator was more resistant to compost treatment than fecal coliforms, with reductions averaging only 1-2.5 logs for enterococci, male-specific coliphages, and sulfite-reducing Clostridia while 5-log reductions were observed for fecal coliforms. Lastly, biosolid isolates from culture-based methods were identified using DNA sequencing-these results revealed that non-enterococci, including Bacillus spp. and Vagococcus spp., were commonly isolated from compost and TPAD biosolids using mEI agar. Given the equivalency of culture- and qPCR-based enterococci concentrations in biosolids and the more conservative inactivation noted for both assays during class A composting, the use of enterococci qPCR monitoring could bypass non-specificity issues with culture-based methods while providing an improved description of pathogen fate in biosolids. PMID:19781735

Viau, Emily; Peccia, Jordan

2009-11-01

259

An ELISA assay for cytochrome P4501A in fish liver cells  

SciTech Connect

An enzyme-linked immunosorbent assay (ELISA) for measuring cytochrome P4501A (CYP1A) expression in vitro in fish hepatoma cells is described. Cells were cultured as monolayers in 96-microwell cell culture plates and exposed to polychlorinated biphenyl (PCB) congeners 77, 105, 153, and 169; 3-methylcholanthrene (3-MC), and {beta}-naphthoflavone (BNF) for 3 d. Relative CYP1A protein content, CYP1A enzymatic activity, and total protein content were determined directly within the wells. At low concentrations of PCB 77, PCB 169, and 3-MC, the ethoxyresorufin-O-deethylase (EROD) activity was induced, but it was inhibited at high concentrations of these compounds. However, CYP1A protein content measured in an ELISA performed with intact cells increased monotonically in response to the concentration. No CYP1A induction was observed for PCB 105 and PCB 153. Because comparison between EROD activity and CYP1A amount gives information about the catalytic efficiency of CYP1A in the cells, this noncompetitive, solid-phase ELISA is recommended as a complementary method to the EROD assay. This novel ELISA method may be an accurate in vitro technique for a rapid and sensitive screening of CYP1A-inducible compounds.

Brueschweiler, B.J.; Fent, K. [Swiss Federal Inst. for Environmental Science and Technology, Duebendorf (Switzerland)]|[Swiss Federal Inst. of Technology Zuerich, Duebendorf (Switzerland); Wuergler, F.E. [Swiss Federal Inst. of Tech., Schwerzenbach (Switzerland). Inst. of Toxicology]|[Univ. of Zuerich, Schwerzenbach (Switzerland)

1996-04-01

260

Comparison of sensitivity to arsenic compounds between a Bhas 42 cell transformation assay and a BALB/c 3T3 cell transformation assay.  

PubMed

A short-term cell transformation assay has recently been developed, using Bhas 42 cells which were established from BALB/c 3T3 cells transfected by v-Ha-ras gene and postulated to be initiated in the two-stage carcinogenesis theory. The Bhas 42 cell transformation assay has been reported to be capable of detecting initiating and promoting activities of chemical carcinogens, according to the different protocols, initiation assay and promotion assay, respectively. The assay is superior to classical transformation assays in cost and labor performance. The present study was carried out to compare its sensitivity with that of a classical BALB/c 3T3 cell system. We performed the Bhas 42 cell transformation assay with inorganic arsenic compounds which are potent environmental carcinogens in human but not mutagens in bacteria or weak mutagens in mammalian cells in vitro. Sodium arsenite, disodium arsenate, and their metabolites, monomethylarsonic acid and dimethylarsinic acid (DMAA) were included in the study. Sodium arsenite was positive in the initiation assay and all compounds except for DMAA were positive in the promotion assay. These results were compared with reported data in a two-stage BALB/c 3T3 cell transformation assay. The sensitivity of Bhas 42 cell transformation assay was found to be similar to that of the conventional BALB/c 3T3 cell transformation assay for the detection of initiating activities of arsenic compounds. For the detection of promoting activities, its sensitivity was equivalent to that of the two-stage BALB/c 3T3 cell transformation assay where the target cells were initiated with sub-threshold dose of 3-methylcholanthrene, confirming that Bhas 42 cells behave as initiated cells in the transformation assay. PMID:19386250

Muramatsu, Dai; Sasaki, Kiyoshi; Kuroda, Sachiko; Hayashi, Kumiko; Tanaka, Noriho; Sakai, Ayako

2009-04-30

261

Ethylene Production by Plant Cell Cultures  

PubMed Central

Suspension cultures of Rosa sp., soybean (Glycine max L.), wheat (Triticum monococcum L.), sweet clover (Melilotus alba Desc.), Haplopappus gracilis Nutt., and rue (Ruta graveolens) produced ethylene. The amount varied with the species. The rate of formation in rose and Haplopappus cells paralleled growth but accelerated when the stationary phase was reached, after which the rate declined sharply. Light was not required for ethylene production. Exogenous ethylene could not replace 2,4-dichlorophenoxyacetic acid or naphthalineacetic acid in the cell cultures, and there was no stimulation of growth in the normal medium. Ethylene at 20 mm reduced growth of Ruta and rose cells by 30 and 20%, respectively. The amounts of ethylene produced by the cultures do not affect growth. Images

Larue, T. A. G.; Gamborg, O. L.

1971-01-01

262

Verbascoside production by plant cell cultures.  

PubMed

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. japonicum f. barbinerve showed rapid growth and the highest yield of verbascoside (1.89 g/l). In these cultures, the effects of major salt concentration in B5 medium on cell growth and verbascoside production were examined. Maximum cell growth and maximum verbascoside production were both achieved by reducing the major salt concentration to half that of the original medium. PMID:24213785

Inagaki, N; Nishimura, H; Okada, M; Mitsuhashi, H

1991-01-01

263

Microfabricated Platforms for Mechanically Dynamic Cell Culture  

PubMed Central

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

Moraes, Christopher; Sun, Yu; Simmons, Craig A.

2010-01-01

264

Biosynthesis of cellulose: studies with tobacco protoplasts and cultured cells  

SciTech Connect

The cell wall of regenerating tobacco protoplasts was shown to be mainly composed of noncellulosic ..beta..-1,3- and ..beta..-1,4-linked glucans with a cellulose content of only about 5%. Some pectic and hemicellulosic material is released by these protoplasts into the culture medium. The DP distribution of the ..cap alpha..-cellulose in regenerating protoplasts as well as in suspension-cultured cells, callus, or tobacco mesophyll revealed the existence of mainly two DP fractions with low (DP<500) and higher (DP 2000-3000) molecular weight, both of which contribute to the cellulosic network of the primary cell wall. The alkali-soluble and alkali-insoluble products of glucan synthetase assays with particulate enzyme fractions were analyzed in detail. By prelabeling with (/sup 14/C)glucose, the existence of primer glucans, which are elongated in the appropriate in vitro assay, could be substantiated. Alkali-soluble glucans consisted of a very short, if any, primer glucan, to which about 40 glucose units were added in vitro. The glucans in the alkali-insoluble fraction have an average DP of 200-250 and are synthesized in vitro by chain elongation via addition of about 30 new glucose units to a 1,4-linked primer glucan of DPapprox.200. 27 references, 6 figures, 2 tables.

Franz, G.; Blaschek, W.; Haass, D.; Koehler, H.

1983-01-01

265

[Further development of a cell culture model for the detection of bacterial pyrogens  

PubMed

In many cases the limulus amoebocyte lysate assay (LAL) is now accepted to detect pyrogenic contaminations in parenteralia instead of the rabbit pyrogenicity test. As the LAL test is able to detect only compounds of gramnegative bacteria (endotoxins) and as it is difficult to test samples with high amounts of protein, we have now further developed our previously established in vitro assay for the detection of bacterial pyrogens in order to overcome the shortcomings of the LAL assay. Our test system is based on the stimulation of IFN-gamma-primed human (THP-1) and murine (J774A.1 and RAW 264.7) monocytic cell lines by bacterial pyrogens to form neopterin or nitrite, respectively. THP-1 in serum-containing media can be used as a high sensitivity assay, while RAW 264.7 in serum-free media is a very good screening system. We showed that the cell-free supernatants of various S. aureus cultures could not be assessed by the LAL assay while the here presented cell culture model detected them very sensitively. The stimulation of immune cells seems to be caused by substances of various molecular masses. In conclusion, we suggest to reduce or even replace the rabbit pyrogenicity test by using our cell culture model in addition to the LAL assay. PMID:11148756

Peterbauer; Werner; Werner-Felmayer

1999-01-01

266

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

267

Comparison of rat and hamster hepatocyte primary culture/DNA repair assays  

SciTech Connect

Previous studies have demonstrated marked differences in the capacity of hepatocytes from rats or hamsters to mediate the metabolic activation of chemical carcinogens to genotoxic (i.e., mutagenic) products. Thus far, very few investigations of species differences in DNA repair have been performed. Therefore, a comparison of the relative extent of DNA repair elicited by various genotoxic chemicals in rat and hamster hepatocyes was conducted, using the hepatocyte primary culture/DNA repair (HPC/DR) assay. Of the ll chemicals tested, eight were more potent in inducing DNA repair in hamster hepatocytes than in rat hepatocytes. Dimethylnitrosamine, diethylnitrosamine, 2-acetylaminofluorene, 9-aminoacridine, pararosaniline hydrochloride, 1-naphthylamine, benzidine and 1,2,3,4-diepoxybutane were all active in hamster hepatocytes at a concentration at least ten times less than the lowest effective concentration in rat hepatocytes. The direct-acting alkylating agent, methylmethane sulfonate, was equipotent inducing DNA repair in both rat and hamster hepatocytes, indicating that the differences in DNA repair observed for the other chemicals were probably not a result of species differences in DNA repair capacities. In contrast, 1-nitropyrene produced a greater DNA repair response in rat hepatocyes than hamster hepatocytes, while the bacterial mutagen 3-(chloromethyl)pyridine hydrochloride was inactive in both hepatocyte systems. These studies demonstrate the feasibility of using hamster hepatocytes in the HPC/DR assay and illustrate the utility of performing the assay with hepatocytes from more than one species.

Kornbrust, D.J.; Barfknect, T.R.

1984-01-01

268

IL-12 elispot assays to detect and enumerate IL-12 secreting cells.  

PubMed

The cytokine IL-12 promotes Th(1)type immune responses and plays a key role in immune regulation. The complex nature of IL-12 hampered its detection without use of stimulants that might give less relevant information. To detect circulating IL-12 p40, we developed enzyme-linked immunospot (ELISPOT) assays that allow enumeration of IL-12 p40 secreting cells without prior in vitro stimulation of the cells. In parallel, intracellular staining of IL-12 p40 by flow cytometry was performed to compare the two methods. IL-12 p40 secreting cells were detected in healthy subjects at a mean number of 103+/-155 per 10(5)blood mononuclear cells (MNC). Numbers of IL-12 p40 secreting blood MNC correlated with IL-12 p40 positive blood MNC detected by flow cytometry. Bacterial endotoxins and the inflammatory cytokines TNF-alpha and IFN-gamma control IL-12 production by antigen presenting cells. Utilizing IL-12 p40 ELISPOT assays, we could confirm occurrence of elevated numbers of IL-12 p40 secreting blood MNC after stimulation with TNF-alpha, IFN-gamma, LPS, LPS+TNF-alpha or LPS+IFN-gamma, compared to cultures without stimulant. Due to its central role in inflammation and autoimmunity, IL-12 is an attractive target for immunotherapy. IL-12 p40 ELISPOT assays represent a sensitive, specific and reliable tool for investigating the role of IL-12 in both health and disease. PMID:10930299

Ozenci, V; Kouwenhoven, M; Press, R; Link, H; Huang, Y M

2000-08-01

269

In vitro genotoxicity assay of sidestream smoke using a human bronchial epithelial cell line.  

PubMed

Genotoxic effects of air contaminants, such as gaseous or particulate compounds, have been difficult to investigate due to inefficient methods for exposing cell cultures directly to these substances. New cultivation and exposure techniques enable treatment of epithelial cells with sample atmospheres with subsequent in vitro assays, as demonstrated by a new system called CULTEX (CULTEX: patent No. DE 19801763; PCT/EP99/00295), which uses a transwell membrane technique for direct exposure of complex mixtures, for example sidestream cigarette smoke, at the air/liquid interface. The sensitivity and susceptibility of human bronchial epithelial cells to this complex mixture have already been shown for cytotoxic endpoints. In this study, genotoxic effects of sidestream cigarette smoke at different concentrations were assessed using the alkaline comet assay. HFBE 21 cells were exposed for 1 h to clean air, nitrogen dioxide or sidestream smoke. Exposure of the cells to sidestream cigarette smoke induced DNA strand breaks in a dose-dependent manner. The combination of gas phase exposure and the comet assay provides a realistic and efficient model for sensitive detection of DNA strand breaks induced by airborne and inhalable compounds. PMID:11983279

Wolz, L; Krause, G; Scherer, G; Aufderheide, M; Mohr, U

2002-06-01

270

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.

Mroue, Rana; Bissell, Mina J.

2013-01-01

271

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.

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

2009-01-01

272

Interaction of Salmonella enterica serovar Typhi with cultured epithelial cells: roles of surface structures in adhesion and invasion  

Microsoft Academic Search

In this study we investigate the ability of Salmonella enterica serovar Typhi (S. Typhi) surface structures to influence invasion and adhesion in epithelial cell assay systems. In general, S. Typhi was found to be less adherent, invasive and cytotoxic than S. enterica serovar Typhimurium (S. Typhimurium). Culture conditions had little effect on adhesion of S. Typhi to cultured cells but

Anne Bishop; D. House; T. Perkins; S. Baker; R. A. Kingsley; G. Dougan

2008-01-01

273

Determining the macropinocytic index of cancer cells through a quantitative image-based assay  

PubMed Central

Macropinocytosis serves as an internalization pathway for extracellular fluid and its contents and is upregulated in oncogene-expressing cells. Recently, we have revealed a functional role for macropinocytosis in fueling cancer cell growth through the internalization of extracellular albumin, which is degraded into a usable source of intracellular amino acids. Assessing macropinocytosis has been challenging in the past due to the lack of reliable assays capable of quantitatively measuring this uptake mechanism. Here, we describe a protocol for visualizing and quantifying the extent of macropinocytosis in cancer cells both in culture and in vivo. Using this approach, the ‘Macropinocytic Index’ of a particular cancer cell line or subcutaneous tumor can be ascertained within 1–2 days. The protocol can be carried out with multiple samples in parallel and can be easily adapted for a variety of cell types and xenograft/allograft mouse models.

Commisso, Cosimo; Flinn, Rory J.; Bar-Sagi, Dafna

2014-01-01

274

Effects of several salt marsh plants on mouse spleen and thymus cell proliferation using mtt assay  

NASA Astrophysics Data System (ADS)

In the present study, we have tested the effects of 21 salt marsh plants on cell proliferation of mouse immune cells (spleen and thymus) using MTT assay in culture. The methanolic extracts of six salt marsh plants ( Rosa rugosa, Ixeris tamagawaensis, Artemisia capillaris, Tetragonia tetragonoides, Erigeron annus, and Glehnia littoralis) showed very powerful suppressive effects of mouse immune cell death and significant activities of cell proliferation in vitro. Especially, the methanolic extract of Rosa rugosa was found to have fifteen times compared to the control treatment, demonstrating that Rosa rugosa may have a potent stimulation effect on immune cell proliferation. These results suggest that several salt marsh plants including Rosa rugosa could be useful for further study as an immunomodulating agent.

Seo, Youngwan; Lee, Hee-Jung; Kim, You Ah; Youn, Hyun Joo; Lee, Burm-Jong

2005-12-01

275

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

USGS Publications Warehouse

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.

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

2013-01-01

276

Functional NK cell cytotoxicity assays against virus infected cells.  

PubMed

Natural Killer (NK) cells are crucial to the control of many viral infections. They are able to kill infected cells directly through the secretion of cytotoxic granules or through binding to death receptors on target cells. They also secrete cytokines and chemokines and, through interactions with dendritic cells, can shape adaptive immunity. The activity of NK cells can be controlled by a balance of activating and inhibitory signals conveyed through ligands on target cells binding to receptors on the NK cell. As a result viruses have devised mechanisms to modulate the expression of NK ligands on target cells, interfering with NK cell recognition and prolonging the life of infected cells. An understanding of how viruses modulate the NK response can lead to an understanding both of NK cell function, and of virus pathogenesis. Measuring the ability of NK cells to kill target cells infected with different viruses, or expressing different viral proteins, is an invaluable technique to identify the proteins and mechanisms by which viruses modulate the NK response. Here we describe two methods to measure this; one method measures sodium dichromate (51)Cr that is released from target cells as they are killed, and the other uses 7-amino-actinomycin D (7-AAD) to measure apoptosis and death of target cells following incubation with NK cells. PMID:23996265

Aicheler, Rebecca J; Stanton, Richard J

2013-01-01

277

Use of the mitochondria toxicity assay for quantifying the viable cell density of microencapsulated jurkat cells.  

PubMed

The mitochondria toxicity assay (MTT assay) is an established method for monitoring cell viability based on mitochondrial activity. Here the MTT assay is proposed for the in situ quantification of the living cell density of microencapsulated Jurkat cells. Three systems were used to encapsulate the cells, namely a membrane consisting of an interpenetrating polyelectrolyte network of sodium cellulose sulphate/poly(diallyldimethylammonium chloride) (NaCS/PDADMAC), a calcium alginate hydrogel covered with poly(L-lysine) (Ca-alg-PLL), and a novel calcium alginate-poly(ethylene glycol) hybrid material (Ca-alg-PEG). MTT results were correlated to data obtained by the trypan blue exclusion assay after release of the cells from the NaCS/PDADMAC and Ca-alg-PLL capsules, while a resazurin-based assay was used for comparison in case of the Ca-alg-PEG material. Analysis by MTT assay allows quick and reliable determination of viable cell densities of encapsulated cells independent of the capsule material. The assay is highly reproducible with inter-assay relative standard deviations below 10%. PMID:23636962

Werner, M; Biss, K; Jérôme, V; Hilbrig, F; Freitag, R; Zambrano, K; Hübner, H; Buchholz, R; Mahou, R; Wandrey, C

2013-01-01

278

Evolution of cell culture systems for HCV.  

PubMed

Many challenges exist for the study of HCV in the laboratory. Therapy using interferon (IFN) is expensive, not well tolerated and ineffective for many patients. HCV research has been hampered by the lack of a robust tissue culture system, but recent advances have made virus growth in culture possible. Cell culture systems using genetically engineered viruses have been reviewed extensively, but here we review recent advances made in the use of natural isolates and the molecular challenges that have been used to overcome the limitations in their growth. Six major genotypes have been identified for HCV that are further divided into numerous subtypes. Combination therapy utilizing IFN-? and ribavirin is the standard of care, but is successful in only one-half of patients. The reasons for IFN resistance may be viral- or host-related and may be due to multiple factors. Recently, telaprevir and boceprevir, together with IFN-? and ribavirin, have been added to the standard of care in patients infected with IFN-resistant genotypes. A major obstacle in the development of effective vaccines and improved therapeutics has been the lack of a reproducible and efficient tissue culture system for propagation of HCV. Many cell culture systems have used genetically-engineered viruses to gain growth in culture through the use of replicons, but recent advances using natural isolates may improve the outlook for progress in HCV research. PMID:23792335

Taylor, Deborah R

2013-01-01

279

Photoreceptor-like cells from reprogramming cultured mammalian RPE cells  

PubMed Central

Purpose Previous studies showed that chick retinal pigment epithelium (RPE) cells can be reprogrammed by a specific gene to take on the path of photoreceptor differentiation. In this study, we tested whether this reprogramming scheme could be applied to mammalian RPE cells. Methods Human RPE cell lines ARPE-19, a spontaneously transformed line of RPE cells derived from a 19-year-old person, and hTERT-RPE1, a telomerase-immortalized RPE cell line derived from a 1-year-old person, were commercially obtained and cultured as recommended. Primary RPE cell cultures were established using RPE isolated from 3- to 6-month-old pig and postnatal day 5 mouse. Cultured cells were transduced with a virus expressing neuroD, neurogenin1 (ngn1), or ngn3, basic helix-loop-helix (bHLH) genes previously identified as capable of inducing RPE-to-photoreceptor reprogramming in the chick system. Alternatively, cells in the culture were transfected chemically or physically through electroporation with vector DNA expressing one of the three genes. The cultures were then analyzed for RPE-to-photoreceptor reprogramming with in situ hybridization and/or immunostaining for photoreceptor gene expression. Results Both hTERT-RPE1 and ARPE-19 cultures gave rise to cells bearing markers of photoreceptors after transduction or transfection with vehicles expressing neuroD or ngn1. The new cells expressed genes encoding photoreceptor proteins, including interphotoreceptor retinoid-binding protein IRBP), recoverin, retinal cone arrestin 3, transducin ?-subunit, Cone-rod homeobox protein (Crx), and red opsin. They displayed morphologies resembling differentiating photoreceptor cells. In primary porcine and mouse RPE cell cultures, transduction with lenti virus (Lvx-IRES-ZsGreen1) expressing ngn1 or ngn3 resulted in the emergence of ZsGreen1+ cells that exhibited morphologies reminiscent of differentiating photoreceptor cells. Immunochemistry showed that some ZsGreen1+ cells were positive for neural marker microtubule-associated protein 2 (Map2) and photoreceptor hallmark proteins red opsin and rhodopsin. Conclusions The results suggest that cells in human RPE cell lines and in primary cultures of porcine and mouse RPE respond to gene-induced reprogramming by giving rise to photoreceptor-like cells. The responsiveness of primary RPE cells, especially those from porcine cells, enhances the biologic feasibility of exploring RPE-to-photoreceptor reprogramming for in situ mammalian photoreceptor replacement without cell transplantation.

Yan, Run-Tao; Huang, Jian; Guidry, Clyde; Wang, Shu-Zhen

2013-01-01

280

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.

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

2011-01-01

281

Fluorescence-based recombination assay for sensitive and specific detection of genotoxic carcinogens in human cells.  

PubMed

In vitro genotoxicity tests are known to suffer from several shortcomings, mammalian cell-based assays, in particular, from low specificities. Following a novel concept of genotoxicity detection, we developed a fluorescence-based method in living human cells. The assay quantifies DNA recombination events triggered by DNA double-strand breaks and damage-induced replication fork stalling predicted to detect a broad spectrum of genotoxic modes of action. To maximize sensitivities, we engineered a DNA substrate encompassing a chemoresponsive element from the human genome. Using this substrate, we screened various human tumor and non-transformed cell types differing in the DNA damage response, which revealed that detection of genotoxic carcinogens was independent of the p53 status but abrogated by apoptosis. Cell types enabling robust and sensitive genotoxicity detection were selected for the generation of reporter clones with chromosomally integrated DNA recombination substrate. Reporter cell lines were scrutinized with 21 compounds, stratified into five sets according to the established categories for identification of carcinogenic compounds: genotoxic carcinogens ("true positives"), non-genotoxic carcinogens, compounds without genotoxic or carcinogenic effect ("true negatives") and non-carcinogenic compounds, which have been reported to induce chromosomal aberrations or mutations in mammalian cell-based assays ("false positives"). Our results document detection of genotoxic carcinogens in independent cell clones and at levels of cellular toxicities <60 % with a sensitivity of >85 %, specificity of ?90 % and detection of false-positive compounds <17 %. Importantly, through testing cyclophosphamide in combination with primary hepatocyte cultures, we additionally provide proof-of-concept for the identification of carcinogens requiring metabolic activation using this novel assay system. PMID:24671466

Ireno, Ivanildce C; Baumann, Cindy; Stöber, Regina; Hengstler, Jan G; Wiesmüller, Lisa

2014-05-01

282

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.

2012-02-01

283

Real-time fluorescence detection of multiple microscale cell culture analog devicesin situ  

Microsoft Academic Search

We investigated multiple microscale cell culture analog (lCCA) assays in situ with a high-throughput imaging system that provides quantitative, nondestructive, and real- time data on cell viability. Since samples do not move between measurements, captured images allow accurate time-course measurements of cell population response and track- ing the fate of each cell type on a quantitative basis. The optical system

Taek-il Oh; Jong Hwan Sung; Daniel A. Tatosian; Michael L. Shuler; Donghyun Kim

2007-01-01

284

High Frequency Retrotransposition in Cultured Mammalian Cells  

Microsoft Academic Search

We previously isolated two human L1 elements (L1.2 and LRE2) as the progenitors of disease-producing insertions. Here, we show these elements can actively retrotranspose in cultured mammalian cells. When stably expressed from an episome in HeLa cells, both elements retrotransposed into a variety of chromosomal locations at a high frequency. The retrotransposed products resembled endogenous L1 insertions, since they were

John V Moran; Susan E Holmes; Thierry P Naas; Ralph J DeBerardinis; Jef D Boeke; Haig H Kazazian

1996-01-01

285

Wound Coverage by Cultured Skin Cells.  

National Technical Information Service (NTIS)

At the conclusion of a three year study on ways to improve wound healing by cultured epidermal grafts, we have found that: We were able to grow epidermal cells on collapsed collagen sponges. As a result, we can create a skin transplant with a quarter of t...

M. Eisinger

1988-01-01

286

Wound Coverage by Cultured Skin Cells.  

National Technical Information Service (NTIS)

The aim was to confirm and extend in vitro and in vivo studies reported last year and develop new means for wound treatment. For the studies in vitro we used different tissue culture techniques. Quantitative data were obtained by cell counts and incorpora...

M. Eisinger

1987-01-01

287

Wound Coverage by Cultured Skin Cells.  

National Technical Information Service (NTIS)

The major purpose of our work is to refine the technology for growth of human epidermal cells to achieve more rapid growth in vitro and easier handling of tissue cultured materials in clinics. It is also to evaluate the possibilities for the use of alloge...

M. Eisinger E. M. Duffy

1985-01-01

288

Neurosphere Culture and Human Organotypic Model to Evaluate Brain Tumor Stem Cells  

PubMed Central

Summary The brain tumor stem cell (BTSC) hypothesis is based on the premise that there is a subpopulation of cells within tumors with tumorigenic and pluripotent properties. BTSC are believed to be responsible for both the initiation of brain tumors and their resistance to current therapeutic modalities. This new paradigm stresses the need for adequate techniques to culture and characterize this special population of cells. Furthermore, the use of different cell migration assays offers the possibility to evaluate the processes involved in glioma metastasis. In this chapter, we summarize a method to culture, analyze the cellular characteristics, and study the invasion of BTSCs using a neurosphere assay, cryostat sectioning, and human organotypic brain cortex migration assay, respectively.

Guerrero-Cazares, Hugo; Chaichana, Kaisorn L.; Quinones-Hinojosa, Alfredo

2010-01-01

289

Prevention and Detection of Mycoplasma Contamination in Cell Culture  

PubMed Central

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

Nikfarjam, Laleh; Farzaneh, Parvaneh

2012-01-01

290

Biphasic Response of Ciprofloxacin in Human Fibroblast Cell Cultures  

PubMed Central

To investigate the possibility of the involvement of an oxidative stress induction in the mechanism of the cytotoxic effect of quinolone antibiotics, we examined the viability of human fibroblast cells exposed to ciprofloxacin (CPFX), and measured the levels of lipid peroxidation (LP), glutathione (GSH), and the activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX). The data showed that the effect of CPFX on the viability of cells, as determined by neutral red uptake assay, was time-dependent, and the dose-response relation was biphasic. Cytotoxicity was not observed in the concentration range 5–150 mg/l CPFX when the cells were incubated for 24 h. In contrast, lower concentrations (5 and 12.5 mg/l) of CPFX increased the cell growth in all incubation periods tested. Marked decreases in the viability of fibroblasts were observed at concentrations 50 and 75 mg/l, and ?50 mg/l, following 48 and 72 h exposure, respectively (p < 0.05). However, when the cells were exposed to > 75 mg/l CPFX for 48 h, no cytotoxicity was observed. By exposing fibroblast cultures to 75 mg/l CPFX for 48 h, an induction of LP enhancement and a marked decrease in intracellular GSH were observed. Vitamin E pretreatment of the cells lowered the level of LP, increased the total GSH content, and provided significant protection against CPFX-induced cytotoxicity. The biphasic effect of CPFX possibly resulted from the complex dose-dependent relationships between reactive oxygen species (ROS), cell proliferation, and cell viability. It was previously reported, in fact, for several cell models that ROS exert a biphasic effect on cell growth. Furthermore, cultured fibroblasts release their own free radicals, and the inhibition of endogenous ROS inhibits the fibroblast cell proliferation, whereas the effect of exogenous ROS is biphasic.

Hincal, Filiz; Gurbay, Aylin; Favier, Alain

2003-01-01

291

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

292

[Cell culture system for hepatitis E virus].  

PubMed

Early studies reported propagation of hepatitis E virus (HEV) in primary hepatocytes or several established cell lines, but replication was inefficient. Recently, using inocula comprised of fecal suspensions with high loads of HEV, originally obtained from Japanese patients who contracted domestic infection of genotype 3 HEV (the JE03-1760F strain, 2.0 x 10(7) copies/ml) or genotype 4 HEV (the HE-JF5/15F strain, 1.3 x 10(7) copies/ml), we developed an efficient cell culture system for HEV in PLC/PRF/5 and A549 cells, which yielded the highest HEV load of 10(8) copies/ml in the culture supernatant, and we successfully propagated six or more generations in serial passages of culture supernatant. In addition, we constructed a full-length infectious cDNA clone (pJE03-1760F/wt) of the JE03-1760F strain, which can replicate efficiently in PLC/PRF/5 and A549 cells. Using a derivative ORF3-deficient (delta ORF3) mutant, we demonstrated that the ORF3 protein of HEV is responsible for virion egress from infected cells and is present on the surface of released HEV particles, which is associated with lipids. Various HEV strains in blood circulation were also propagated efficiently in PLC/PRF/5 and A549 cells. Our in vitro cell culture system can be used for propagation of a wide variety of HEV strains in feces and sera from various infected patients, allowing extended studies on viral replication specific to different HEV strains. PMID:20848869

Okamoto, Hiroaki

2010-06-01

293

Efficiency of embryoid body formation and hematopoietic development from embryonic stem cells in different culture systems.  

PubMed

Embryonic stem (ES) cells have tremendous potential as a cell source for cell-based therapies. Realization of that potential will depend on our ability to understand and manipulate the factors that influence cell fate decisions and to develop scalable methods of cell production. We compared four standard ES cell differentiation culture systems by measuring aspects of embryoid body (EB) formation efficiency and cell proliferation, and by tracking development of a specific differentiated tissue type-blood-using functional (colony-forming cell) and phenotypic (Flk-1 and CD34 expression) assays. We report that individual murine ES cells form EBs with an efficiency of 42 +/- 9%, but this value is rarely obtained because of EB aggregation-a process whereby two or more individual ES cells or EBs fuse to form a single, larger cell aggregate. Regardless of whether EBs were generated from a single ES cell in methylcellulose or liquid suspension culture, or aggregates of ES cells in hanging drop culture, they grew to a similar maximum cell number of 28,000 +/- 9,000 cells per EB. Among the three methods for EB generation in suspension culture there were no differences in the kinetics or frequency of hematopoietic development. Thus, initiating EBs with a single ES cell and preventing EB aggregation should allow for maximum yield of differentiated cells in the EB system. EB differentiation cultures were also compared to attached differentiation culture using the same outputs. Attached colonies were not similarly limited in cell number; however, hematopoietic development in attached culture was impaired. The percentage of early Flk-1 and CD34 expressing cells was dramatically lower than in EBs cultured in suspension, whereas hematopoietic colony formation was almost completely inhibited. These results provide a foundation for development of efficient, scalable bioprocesses for ES cell differentiation, and inform novel methods for the production of hematopoietic tissues. PMID:11948451

Dang, Stephen M; Kyba, Michael; Perlingeiro, Rita; Daley, George Q; Zandstra, Peter W

2002-05-20

294

Stem cell factor amplifies newborn and sickle erythropoiesis in liquid cultures.  

PubMed

A two-phase liquid-culture system was used to substantially amplify and differentiate erythroblasts, starting with mononuclear cells from the blood of normal adults, newborn infants, and patients with sickle cell anemia. After the first 7 days (phase 1), in medium plus fetal bovine serum (FBS) alone, or in combination with stem cell factor (SCF) or conditioned medium (CM), the cell number was unchanged, and the cells all looked like lymphocytes. These cells were then diluted into medium with erythropoietin (Ep) alone, with Ep and either SCF or CM, or in methylcellulose with the same factors (phase 2). After 14 days in liquid phase 2 with SCF and Ep, the cell numbers increased an average of 30-fold in the sickle, 24-fold in the newborn, and 4-fold in the normal adult cultures; almost all the cells were erythroblasts and erythrocytes. SCF in phase 1 increased the number of late progenitors (CFU-E) assayed in methylcellulose, with the largest number in sickle, followed by newborn cultures and then adult cultures. We conclude that erythroid progenitor cells survive for at least 7 days without Ep (but with FBS). Progenitor cells are amplified, particularly with SCF. Later in culture, SCF with Ep increases the final number of differentiated erythroid cells. Both the early and the late effects of SCF are most effective in sickle, followed by newborn cultures and then adult cultures. PMID:7683924

Weinberg, R S; Thomson, J C; Lao, R; Chen, G; Alter, B P

1993-05-15

295

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

PubMed Central

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

2011-01-01

296

Computerized microfluidic cell culture using elastomeric channels and Braille displays  

PubMed Central

Computer-controlled microfluidics would advance many types of cellular assays and microscale tissue engineering studies wherever spatiotemporal changes in fluidics need to be defined. However, this goal has been elusive because of the limited availability of integrated, programmable pumps and valves. This paper demonstrates how a refreshable Braille display, with its grid of 320 vertically moving pins, can power integrated pumps and valves through localized deformations of channel networks within elastic silicone rubber. The resulting computerized fluidic control is able to switch among: (i) rapid and efficient mixing between streams, (ii) multiple laminar flows with minimal mixing between streams, and (iii) segmented plug-flow of immiscible fluids within the same channel architecture. The same control method is used to precisely seed cells, compartmentalize them into distinct subpopulations through channel reconfiguration, and culture each cell subpopulation for up to 3 weeks under perfusion. These reliable microscale cell cultures showed gradients of cellular behavior from C2C12 myoblasts along channel lengths, as well as differences in cell density of undifferentiated myoblasts and differentiation patterns, both programmable through different flow rates of serum-containing media. This technology will allow future microscale tissue or cell studies to be more accessible, especially for high-throughput, complex, and long-term experiments. The microfluidic actuation method described is versatile and computer programmable, yet simple, well packaged, and portable enough for personal use.

Gu, Wei; Zhu, Xiaoyue; Futai, Nobuyuki; Cho, Brenda S.; Takayama, Shuichi

2004-01-01

297

Computerized microfluidic cell culture using elastomeric channels and Braille displays.  

PubMed

Computer-controlled microfluidics would advance many types of cellular assays and microscale tissue engineering studies wherever spatiotemporal changes in fluidics need to be defined. However, this goal has been elusive because of the limited availability of integrated, programmable pumps and valves. This paper demonstrates how a refreshable Braille display, with its grid of 320 vertically moving pins, can power integrated pumps and valves through localized deformations of channel networks within elastic silicone rubber. The resulting computerized fluidic control is able to switch among: (i) rapid and efficient mixing between streams, (ii) multiple laminar flows with minimal mixing between streams, and (iii) segmented plug-flow of immiscible fluids within the same channel architecture. The same control method is used to precisely seed cells, compartmentalize them into distinct subpopulations through channel reconfiguration, and culture each cell subpopulation for up to 3 weeks under perfusion. These reliable microscale cell cultures showed gradients of cellular behavior from C2C12 myoblasts along channel lengths, as well as differences in cell density of undifferentiated myoblasts and differentiation patterns, both programmable through different flow rates of serum-containing media. This technology will allow future microscale tissue or cell studies to be more accessible, especially for high-throughput, complex, and long-term experiments. The microfluidic actuation method described is versatile and computer programmable, yet simple, well packaged, and portable enough for personal use. PMID:15514025

Gu, Wei; Zhu, Xiaoyue; Futai, Nobuyuki; Cho, Brenda S; Takayama, Shuichi

2004-11-01

298

Automated reagent-dispensing system for microfluidic cell biology assays.  

PubMed

Microscale systems that enable measurements of oncological phenomena at the single-cell level have a great capacity to improve therapeutic strategies and diagnostics. Such measurements can reveal unprecedented insights into cellular heterogeneity and its implications into the progression and treatment of complicated cellular disease processes such as those found in cancer. We describe a novel fluid-delivery platform to interface with low-cost microfluidic chips containing arrays of microchambers. Using multiple pairs of needles to aspirate and dispense reagents, the platform enables automated coating of chambers, loading of cells, and treatment with growth media or other agents (e.g., drugs, fixatives, membrane permeabilizers, washes, stains, etc.). The chips can be quantitatively assayed using standard fluorescence-based immunocytochemistry, microscopy, and image analysis tools, to determine, for example, drug response based on differences in protein expression and/or activation of cellular targets on an individual-cell level. In general, automation of fluid and cell handling increases repeatability, eliminates human error, and enables increased throughput, especially for sophisticated, multistep assays such as multiparameter quantitative immunocytochemistry. We report the design of the automated platform and compare several aspects of its performance to manually-loaded microfluidic chips. PMID:24051515

Ly, Jimmy; Masterman-Smith, Michael; Ramakrishnan, Ravichandran; Sun, Jing; Kokubun, Brent; van Dam, R Michael

2013-12-01

299

Phytotoxicity of Fusarium solani culture filtrates from soybeans and other hosts assayed by stem cuttings  

Microsoft Academic Search

Fusarium solani infects roots of a number of different plant species and some strains produce Phytotoxins. F. solani f. sp. glycines, the causal organism of sudden death syndrome (SDS) of soybean (Glycine max), colonises soybean roots and produces toxin(s) that are translocated to leaves and cause intervienal chlorosis and necrosis.\\u000a Several experiments evaluated the phytotoxicity of cell-free culture filtrates of

G. L. Hartman; Y. H. Huang; S. Li

2004-01-01

300

Assaying stem cell mechanobiology on microfabricated elastomeric substrates with geometrically modulated rigidity.  

PubMed

We describe the use of a microfabricated cell culture substrate, consisting of a uniform array of closely spaced, vertical, elastomeric microposts, to study the effects of substrate rigidity on cell function. Elastomeric micropost substrates are micromolded from silicon masters comprised of microposts of different heights to yield substrates of different rigidities. The tips of the elastomeric microposts are functionalized with extracellular matrix through microcontact printing to promote cell adhesion. These substrates, therefore, present the same topographical cues to adherent cells while varying substrate rigidity only through manipulation of micropost height. This protocol describes how to fabricate the silicon micropost array masters (~2 weeks to complete) and elastomeric substrates (3 d), as well as how to perform cell culture experiments (1-14 d), immunofluorescence imaging (2 d), traction force analysis (2 d) and stem cell differentiation assays (1 d) on these substrates in order to examine the effect of substrate rigidity on stem cell morphology, traction force generation, focal adhesion organization and differentiation. PMID:21293460

Yang, Michael T; Fu, Jianping; Wang, Yang-Kao; Desai, Ravi A; Chen, Christopher S

2011-02-01

301

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

302

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

303

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

304

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

305

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

306

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

307

Establishment and characterization of two cell lines derived from primary cultures of Gekko japonicus cerebral cortex.  

PubMed

Adult Gekko japonicus is one of those vertebrates that are able to regenerate their missing or amputated tail. The most interesting feature of this animal lies in the ability of its spinal cord to regrow a functional tail. A fundamental question is whether the neuroglial cells play a different role compared with high vertebrates. Since in vitro studies using primary neuroglial cells are hampered by the limited lifespan and miscellaneous genetic background of these cells, we generated neuroglial cell lines from primary cell cultures of cerebral cortex of G. japonicus. The SV40 (simian-virus-40) T antigen gene was introduced into primary cell cultures. Cell cycle analysis, cell growth and proliferation, cell colony formation and contact inhibition, as well as karyotype assays were investigated. Two cell colonies, Gsn-1 and Gsn-3, were immunochemically characterized as glial fibrillary acidic protein and galactocerebroside-positive respectively. Compared with parental primary cells, the Gsn cells displayed shorter population doubling time, decreased percentage of cells in the G0/G1 phase, higher cell proliferation index, and increased cell activity. In assays of colony characteristics, Gsn cells showed increased cell activity at the lower cell densities or FBS (fetal bovine serum) supplement. The karyotype of immortalized Gsn cells exhibited transformational characteristics with hyperdiploid and polyploid chromosomes. The cell lines will provide a useful in vitro model for gecko neuroglial cells and facilitate systematic studies investigating the biological functions of specific gene products related to regeneration of the central nervous system. PMID:19947933

Liu, Mei; Gu, Yun; Liu, Yan; Li, Jing; He, Jianghong; Lin, Sheyu; Gu, Xiaosong

2010-02-01

308

Human Sperm Bioassay for Reprotoxicity Testing in Embryo Culture Media: Some Practical Considerations in Reducing the Assay Time  

PubMed Central

Human sperm assay (HSA) is a preferred in house quality control and proficiency test (PT) practiced in fertility laboratories. HSA is performed over varying durations, apparently without following set criteria. To better understand the assay time required for reprotoxicity testing in embryo culture media, we compared American-Association-of-Bioanalysts-(AAB-) administered HSA data to our own assay performed using PT samples obtained from AAB. Participating laboratories were required to culture sperm for 48 hours to determine media acceptability. Conclusions drawn from 48- and 24-hour observations were the same, suggesting that HSA could identify reprotoxic media in less time than required by AAB. Our assay revealed that changes in motility grade in adulterated media are significantly different from those in control media. Furthermore, grade changes can be identified earlier than differences in motility loss between samples. Analyzing motility and motility quality together provides a method for establishing an optimal time for HSA.

Hossain, Amjad; Aryal, Subhash; Osuampke, Collin; Phelps, John

2010-01-01

309

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

PubMed

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

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

2013-12-01

310

Toxicity of copper-based dental alloys in cell culture.  

PubMed

The biocompatibility of three commercial copper-based dental casting alloys--Duracast MS, Goldent, and Trindium--three experimental copper alloys, and a control gold alloy, Modulay, were investigated. Trindium, Duracast MS, experimental alloys 1 and 2 are aluminum bronzes; Goldent is a hybrid aluminum-brass alloy; and experimental alloy #3 is a high zinc brass alloy. ASTM F813-83 Standard Practice for Direct Contact Cell Culture Evaluation of Materials for Medical Devices, a 3-day direct-contact cell culture regimen and atomic absorption spectroscopy were utilized for evaluating the biocompatibility of these alloys in both Waymouth's and RPMI 1640 complete media. Cellular proliferation assays, using 3H-thymidine uptake, were also conducted in Waymouth's media. In this investigation, only the experimental alloy #3 elicited alterations in morphology and viability of the fibroblast monolayer during the ASTM and 3-day culture tests in either media. Cell cultures exposed to experimental alloy #3 experienced copper concentrations greater than 16.0 ppm in Waymouth's and 10 ppm copper in RPMI 1640 media. Differences in the size of the cytotoxic zone around experimental alloy #3 were also observed, with the larger zone occurring in Waymouth's media. In contrast to the direct cell contact studies, all alloys caused decreases in 3H-thymidine uptake in Waymouth's media at much reduced metal ion concentrations as compared to the controls. Thus, adverse changes in DNA synthesis occurred at much lower copper and zinc concentrations than changes in morphology and viability. Consequently, the assessment of biocompatibility is dependent on the parameters evaluated, and several parameters must be analyzed before a material may be considered biocompatible. PMID:2808459

Bumgardner, J D; Lucas, L C; Tilden, A B

1989-10-01

311

An adaptation of the human HepaRG cells to the in vitro micronucleus assay.  

PubMed

The in vitro micronucleus test is considered as an attractive tool for genotoxicity testing of chemicals because of its simplicity of scoring and wide applicability in different cell types. However, most of the cells currently in use are devoid of the enzyme equipment required for activation of promutagens in the genotoxic metabolites. We postulated that the human HepaRG cell line, which can express xenobiotic metabolising enzymes at levels close to those found in primary human hepatocytes and has retained the indefinite growth capacity of transformed cells, could represent a more suitable model for genotoxicity testing of chemicals requiring metabolic activation. Based on the recommendations of the Organisation for Economic Co-operation and Development test guideline TG 487 for testing of chemicals, HepaRG cell cultures containing >80% mature hepatocytes were treated in situ with various chemicals for 24 h followed by a 3-day mitogenic stimulation with epidermal growth factor without cytokinesis block. In such culture conditions, HepaRG cells underwent >1.5 cell cycle per cell during the mitogenic stimulation. While non-genotoxic compounds (mannitol and staurosporine) did not increase the rate of micronucleated mononucleated cells, all aneugens (colchicine, nocodazole and dichlorodiphenyldichloroethylene) as well as the direct acting clastogen methyl methanesulfonate and clastogens requiring metabolic activation (aflatoxin B1, benzo(a)pyrene and 2-nitrofluorene) induced a statistically significant concentration-related increase in the number of mono-micronucleated cells. The micronucleus test was also performed after 7-day repeat exposure of HepaRG cells to the chemicals. Noticeably, a time-dependent effect was obtained with the three clastogens requiring metabolic activation. In conclusion, our results obtained with HepaRG hepatocytes exposed to various genotoxic compounds requiring or not bioactivation, compared favorably with those reported in various other cell types. They support the view that metabolically competent HepaRG cells have unique potential benefits for testing genotoxic compounds using the in vitro micronucleus assay. PMID:22058015

Jossé, Rozenn; Rogue, Alexandra; Lorge, Elisabeth; Guillouzo, André

2012-05-01

312

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

Microsoft Academic Search

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

Izhar U. H. Khan; Jagjit S. Yadav

2004-01-01

313

Enzyme-linked immunosorbent assay using monoclonal antibodies for identification of mycobacteria from early cultures.  

PubMed Central

A simple enzyme-linked immunosorbent assay (ELISA) for the identification of cultured mycobacteria belonging to the Mycobacterium tuberculosis complex, the Mycobacterium avium complex, and Mycobacterium kansasii has been developed (R. Schöningh, C. P. H. J. Verstijnen, S. Kuijper, and A. H. J. Kolk. J. Clin. Microbiol. 28:708-713, 1990). The test for the routine identification of cultured mycobacteria was introduced in five clinical laboratories located in Tanzania, Thailand, Vietnam, and The Netherlands. The ELISA can be conducted without an ELISA reader since the test can be read visually. The results of identification of 255 strains of the M. tuberculosis complex by microbiological means and by ELISA were compared; the specificity and the sensitivity were 100%. For the M. avium complex, the specificity was 100% and the sensitivity was 64%. All 26 M. kansasii strains tested could be identified as M. kansasii. The ELISA described here proved to be useful in both well- and modestly equipped laboratories and may replace the microbiological method of identification of M. tuberculosis and M. kansasii.

Verstijnen, C P; Ly, H M; Polman, K; Richter, C; Smits, S P; Maselle, S Y; Peerbooms, P; Rienthong, D; Montreewasuwat, N; Koanjanart, S

1991-01-01

314

Genomics in mammalian cell culture bioprocessing  

PubMed Central

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

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

2013-01-01

315

Rotavirus-induced fusion from without in tissue culture cells.  

PubMed Central

We present the first evidence of fusion from without induced in tissue culture cells by a nonenveloped virus. Electron micrographs of two strains of rotavirus, bovine rotavirus C486 and rhesus rotavirus, show that virally mediated cell-cell fusion occurs within 1 h postinfection. Trypsin activation is necessary for rotavirus to mediate cell-cell fusion. The extent of fusion is relative to the amount of virus used, and maximum fusion occurs between pHs 6.5 and 7.5. Fusion does not require virus-induced protein synthesis, as virus from both an empty capsid preparation and from an EDTA-treated preparation, which is noninfectious, can induce fusion. Incubation of rotavirus with neutralizing and nonneutralizing monoclonal antibodies before addition to cells indicates that viral protein 4 (VP4; in the form of VP5* and VP8*) and VP7 are involved in fusion. Light and electron micrographs document this fusion, including the formation of pores or channels between adjacent fused cells. These data support direct membrane penetration as a possible route of infection. Moreover, the assay should be useful in determining the mechanisms of cell entry by rotavirus.

Falconer, M M; Gilbert, J M; Roper, A M; Greenberg, H B; Gavora, J S

1995-01-01

316

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

SciTech Connect

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

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

1985-01-01

317

Plant cell cultures: Chemical factories of secondary metabolites  

Microsoft Academic Search

This review deals with the production of high-value secondary metabolites including pharmaceuticals and food additives through plant cell cultures, shoot cultures, root cultures and transgenic roots obtained through biotechnological means. Plant cell and transgenic hairy root cultures are promising potential alternative sources for the production of high-value secondary metabolites of industrial importance. Recent developments in transgenic research have opened up

S Ramachandra Rao; G. A Ravishankar

2002-01-01

318

Cysteine Metabolism in Cultured Tobacco Cells  

PubMed Central

Transported l-[35S]cysteine was rapidly metabolized by cultured tobacco cells when supplied to the cells at 0.02 millimolar or 0.5 millimolar. The internal cysteine pool was expandable to approximately 2400 nmoles per gram fresh weight. The 35S label derived from cysteine was found in several metabolites. The amount of label in glutathione and sulfate was directly proportional to the internal l-[35S]cysteine, while the levels of labeled methionine and protein were apparently independent of internal labeled cysteine. Cysteine was more rapidly metabolized when the external cysteine concentration was low (0.02 millimolar) with up to 90% of the 35S label present as compounds other than cysteine. The initial step in cysteine degradation yielded pyruvate, sulfide, and presumably NH4+. Stoichiometry studies using extracts prepared from acetone powders of tobacco cells indicated that pyruvate and sulfide were produced in a 1:1 ratio. The catabolic reaction was linear with respect to time and amount of protein and had a pH optimum of 8 in crude extracts. Preliminary kinetic data indicated the Km to be approximately 0.2 millimolar. The extractable degradative activity was enhanced 15- to 20-fold by preincubating the cells for 24 hours in 0.5 millimolar cysteine. The extractable specific enzyme activity roughly reflected the growth curve of the cells in culture. Maximal cysteine degradation was observed in extracts prepared from late log phase cultures that were preincubated in cysteine, while little activity was found in similar extracts from stationary phase cultures. These results are consistent with an inducible catabolic enzyme similar to the cysteine desulfhydrase from bacteria.

Harrington, H. Michael; Smith, Ivan K.

1980-01-01

319

Real-time analysis of endosomal lipid transport by live cell scintillation proximity assay  

PubMed Central

A scintillation proximity assay has been developed to study the endosomal trafficking of radiolabeled cholesterol in living cells. Mouse macrophages were cultured in the presence of tritiated cholesterol and scintillant microspheres. Microspheres were taken up by phagocytosis and stored in phagolysosomes. Absorption of tritium ? particles by the scintillant produces light signals that can be measured in standard scintillation counters. Because of the short range of tritium ? particles and for geometric reasons, scintillant microspheres detect only that fraction of tritiated cholesterol localized inside phagolysosomes or within a distance of ~600 nm. By incubating cultures in a temperature-controlled microplate reader, the kinetics of phagocytosis and cholesterol transport could be analyzed in near-real time. Scintillation signals were significantly increased in response to inhibitors of lysosomal cholesterol export. This method should prove a useful new tool for the study of endosomal trafficking of lipids and other molecules.

Stockinger, Walter; Castoreno, Adam B.; Wang, Yan; Pagnon, Joanne C.; Nohturfft, Axel

2007-01-01

320

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

PubMed Central

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

2013-01-01

321

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.

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

322

Patterning of polymeric cell culture substrates.  

PubMed

The purpose of this chapter is to provide a summary of polymer patterning technologies for biological applications and detailed instructions for resist-free deep ultraviolet (UV) patterning of poly(styrene). Photochemical modifications of this polymer yield unstable peroxides together with stable oxidized chemical groups. The altered physicochemical properties of the polymer surface influence protein adsorption and cell adhesion. HepG2 (human hepatoma cell line), fibroblasts (L929, murine fibroblast line), and other cell lines exhibit strong adhesion on areas of UV-irradiated polymer. Masked irradiations open a simple, fast (cell patterns are obtained within a few hours), and economical route to obtain chemically patterned cell culture substrates. The described protocol is advantageous compared to silane-based patterning techniques on glass or thiol-based patterning on gold because of the elimination of any chemical treatment and the small size of achieved structures. The protocol is compatible with common clean room technologies; however, even without access to a clean room, structured substrates can be produced. The described technique can be a useful tool for a variety of cell cultures used to study biological processes like intercellular communication and organogenesis and for applications like biosensing or tissue engineering. PMID:24439278

Welle, Alexander; Weigel, Simone; Bulut, Özgül Demir

2014-01-01

323

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.

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

2014-01-01

324

Cell culture metabolomics in the diagnosis of lung cancer-the influence of cell culture conditions.  

PubMed

Lung cancer is the leading cause of cancer deaths. Unfortunately, lung cancer is often diagnosed only when it becomes symptomatic or at an advanced stage when few treatment options are available. Hence, a diagnostic test suitable for screening widespread populations is required to enable earlier diagnosis. Analysis of exhaled breath provides a non-invasive method for early detection of lung cancer. Analysis of volatile organic compounds (VOCs) by various mass spectral techniques has identified potential biomarkers of disease. Nevertheless, the metabolic origins and the disease specificity of VOCs need further elucidation. Cell culture metabolomics can be used as a bottom-up approach to identify biomarkers of pathological conditions and can also be used to study the metabolic pathways that produce such compounds. This paper summarizes the current knowledge of lung cancer biomarkers in exhaled breath and emphasizes the critical role of cell culture conditions in determining the VOCs produced in vitro. Hypoxic culture conditions more closely mimic the conditions of cancer cell growth in vivo. We propose that since hypoxia influences cell metabolism and so potentially the VOCs that the cancer cells produce, the cell culture metabolomics projects should consider culturing cancer cells in hypoxic conditions. PMID:24861817

Kalluri, U; Naiker, M; Myers, M A

2014-06-01

325

Evaluation of a recombinant yeast cell estrogen screening assay.  

PubMed Central

A wide range of chemicals with diverse structures derived from plant and environmental origins are reported to have hormonal activity. The potential for appreciable exposure of humans to such substances prompts the need to develop sensitive screening methods to quantitate and evaluate the risk to the public. Yeast cells transformed with plasmids encoding the human estrogen receptor and an estrogen responsive promoter linked to a reporter gene were evaluated for screening compounds for estrogenic activity. Relative sensitivity to estrogens was evaluated by reference to 17 beta-estradiol (E2) calibration curves derived using the recombinant yeast cells, MCF-7 human breast cancer cells, and a prepubertal mouse uterotrophic bioassay. The recombinant yeast cell bioassay (RCBA) was approximately two and five orders of magnitude more sensitive to E2 than MCF-7 cells and the uterotrophic assay, respectively. The estrogenic potency of 53 chemicals, including steroid hormones, synthetic estrogens, environmental pollutants, and phytoestrogens, was measured using the RCBA. Potency values produced with the RCBA relative to E2 (100) included estrone (9.6), diethylstilbestrol (74.3), tamoxifen (0.0047), alpha-zearalanol (1.3), equol (0.085), 4-nonylphenol (0.005), and butylbenzyl phathalate (0.0004), which were similar to literature values but generally higher than those produced by the uterotrophic assay. Exquisite sensitivity, absence of test compound biotransformation, ease of use, and the possibility of measuring antiestrogenic activity are important attributes that argue for the suitability of the RCBA in screening for potential xenoestrogens to evaluate risk to humans, wildlife, and the environment. Images Figure 1. Figure 2. Figure 3. Figure 4.

Coldham, N G; Dave, M; Sivapathasundaram, S; McDonnell, D P; Connor, C; Sauer, M J

1997-01-01

326

Neonatal Rat Heart Cells Cultured in Simulated Microgravity.  

National Technical Information Service (NTIS)

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 cel...

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

1994-01-01

327

A rapid imageable in vivo metastasis assay for circulating tumor cells.  

PubMed

Circulating tumor cells (CTCs) are of great importance for cancer diagnosis, prognosis and treatment. It is necessary to improve the ability to image and analyze them for their biological properties which determine their behavior in the patient. In the present study, using immunomagnetic beads, CTCs were rapidly isolated from the circulation of mice orthotopically implanted with human PC-3 prostate cancer cells stably expressing green fluorescent protein (GFP). The PC-3-GFP CTCs were then expanded in culture in parallel with the parental PC-3-GFP cell line. Both cell types were then inoculated onto the chorioallentoic membrane (CAM) of chick embryos. Eight days later, embryos were harvested and the brains were processed for frozen sections. The IV-100 intravital laser scanning microscope enabled rapid identification of fluorescent metastatic foci within the chick embryonic brain. Inoculation of embryos with PC-3-GFP CTCs resulted in a 3 to 10-fold increase in brain metastasis when compared to those with the parental PC-3-GFP cells (p<0.05 in all animals). Thus, PC-3-GFP CTCs have increased metastatic potential compared to their parental counterparts. Furthermore, the chick embryo represents a rapid, sensitive, imageable assay of metastatic potential for CTCs. The chick embryo assay has future clinical application for individualizing patient therapy based on the metastatic profile of their CTCs. PMID:21965717

Menen, Rhiana S; Pinney, Emmett; Kolostova, Katerina; Bobek, Vladimir; Suetsugu, Atsushi; Zhang, Nan; Bouvet, Michael; Hoffman, Robert M

2011-10-01

328

DNA microfiltration assay: a simple technique for detecting DNA damage in mammalian cells.  

PubMed

A simple method for detection of DNA single-strand breaks (DNA-SSB) in cultured cells is described, based on filtration of alkaline-lysed cells through microfilters. After exposure to potentially DNA damaging agents, the cells are transferred to 0.8 micron cellulose acetate filters mounted in microfilter devices where they are washed, lysed and centrifuged to separate undamaged DNA from damaged DNA. When human bronchiolar cells (14Br) were exposed to different DNA damaging agents, hydrogen peroxide, N-methyl-N-nitro-N-nitrosoguanidine or 4-nitroquinoline-1-oxide, there was good correlation between the extent of DNA damage assessed by this filtration technique and by DNA precipitation assay. DNA-SSB were also detected by the filtration technique after exposure of bronchiolar cells to phorbol ester-stimulated human neutrophils. The filtration assay is easy to perform, the sample handling capacity is very high, and no expensive or complicated laboratory equipment is required. It may therefore be an alternative, or a complement, to other methods for detection of DNA-SSB. PMID:8293540

Leanderson, P; Wennerberg, K; Tagesson, C

1994-01-01

329

Apparent functional independence of the mitochondrial and nuclear transcription systems in cultured human cells  

Microsoft Academic Search

We have constructed a series of reporter constructs which test the effects of sequence elements from the control region of human mitochondrial DNA on expression in the nucleus, as assayed by transient expression in cultured human cells. The mitochondrial heavy-strand promoter (HSP) was unable to function as a promoter in nuclear DNA. Neither the HSP, nor the binding region for

Richard Sewards; Bryony Wiseman; Howard T. Jacobs

1994-01-01

330

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

EPA Science Inventory

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

331

Adipose derived mesenchymal stem cells partially rescue mitomycin C treated ARPE19 cells from death in co-culture condition.  

PubMed

Age-related macular degeneration is a retinal disease with important damage at the RPE layer. This layer is considered a target for therapeutical approaches. Stem cell transplantation is a promising option for retinal diseases. Adipose derived mesenchymal stem cells secret growth factors which might play a significant role in RPE maintenance. This study aimed to evaluate human AD-MSCs ability to rescue mitomycin C treated dying ARPE19 cells in co-culture condition. ARPE19 cells were treated with MMC (50 ?g/ml, 100 ?g/ml and 200 ?g/ml) for 2 hours to induce cell death. These treated cells were co-cultured with hAD-MSCs in indirect co-culture system for 3 days and 3 weeks. Then the viability, growth and proliferation of these ARPE19 cells were evaluated by a cell viability/cytotoxicity assay kit and Alamar Blue (AB) assay. Untreated ARPE19 cells and human skin fibroblasts (HSF) were used as controls. MMC blocked ARPE19 cell proliferation significantly in 3 days and cells were almost completely dead after 3 weeks. Cell toxicity of MMC increased significantly with concentration. When these cells were co-cultured with hAD-MSCs, a significant growth difference was observed in treated cells compared to untreated cells. hAD-MSCs rescue capacity was also significantly higher than HSF for treated ARPE19 cells. This study showed that hAD-MSCs rescued MMC treated ARPE19 cells from death. It probably occurred due to undefined growth factors secreted by hAD-MSCs in the medium, shared by treated ARPE19 cells in co-culture conditions. This study supports further evaluation of the effect of hAD-MSCs subretinal transplantation over the RPE degeneration process in AMD patients. PMID:23719745

Singh, Amar K; Srivastava, Girish K; García-Gutiérrez, María T; Pastor, J Carlos

2013-12-01

332

Effect of antioxidants on PDT treatment of cultured tumor cells  

NASA Astrophysics Data System (ADS)

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

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

1998-05-01

333

Insulin receptors in cultured mouse retinal cells  

Microsoft Academic Search

Summary  The binding of125I-insulin to uncloned and cloned cultures of mouse retinal cells has been investigated. At 15° C, binding of the hormone reached a steady state by 60 min, while at 37° C equilibrium was reached earlier but at a lower level than at 15° C. Porcine insulin, porcine proinsulin and guinea pig insulin displaced labelled insulin in proportion to

P. Thomopoulos; B. Pessac

1979-01-01

334

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

335

Determining Cell Number During Cell Culture using the Scepter Cell Counter  

PubMed Central

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses. Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed1 who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate2. To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry1. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments1. The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection3 in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.

Ongena, Kathleen; Das, Chandreyee; Smith, Janet L.; Gil, Sonia; Johnston, Grace

2010-01-01

336

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

337

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

338

Advances in microfluidic cell culture systems for studying angiogenesis.  

PubMed

Angiogenesis, the formation of new blood vessels in the vasculature, is a major research topic in biology with implications in development, cancer, tissue engineering, and regenerative medicine. Although much knowledge has been acquired over many decades through application of various angiogenesis assays, these methods have various drawbacks that limit their overall utility. Given the importance of angiogenesis in our understanding of numerous biological processes and its potential as a therapeutic target in cancer and other diseases, there is need to develop useful tools with improved physiological relevance, accessibility, robustness, and throughput over existing assays. Recent developments in microfluidics have demonstrated enormous potential of microscale cell culture systems for biology studies, especially angiogenesis. This area is advancing rapidly, and it is important to remain up to date with the state of the art in technology and evaluate its current and future impact on angiogenesis research. This review examines the latest advances in microfluidic angiogenesis systems. Design and methodology of microscale systems are discussed, and biological insights obtained from the systems are examined. Importantly, physiological relevance, accessibility, and data output of microfluidic angiogenesis systems are compared with traditional angiogenesis assays, and next challenges facing researchers are presented with consideration of the potential integration of automated systems. PMID:23832929

Young, Edmond W K

2013-12-01

339

Cell-free NADPH oxidase activation assays: "in vitro veritas".  

PubMed

The superoxide (O2 (?-))-generating NADPH oxidase complex of phagocytes comprises a membrane-imbedded heterodimeric flavocytochrome, known as cytochrome b 558 (consisting of Nox2 and p22 (phox) ) and four cytosolic regulatory proteins, p47 (phox) , p67 (phox) , p40 (phox) , and the small GTPase Rac. Under physiological conditions, in the resting phagocyte, O2 (?-) generation is initiated by engagement of membrane receptors by a variety of stimuli, followed by specific signal transduction sequences leading to the translocation of the cytosolic components to the membrane and their association with the cytochrome. A consequent conformational change in Nox2 initiates the electron "flow" along a redox gradient, from NADPH to oxygen, leading to the one-electron reduction of molecular oxygen to O2 (?-). Methodological difficulties in the dissection of this complex mechanism led to the design "cell-free" systems (also known as "broken cells" or in vitro systems). In these, membrane receptor stimulation and all or part of the signal transduction sequence are missing, the accent being placed on the actual process of "NADPH oxidase assembly," thus on the formation of the complex between cytochrome b 558 and the cytosolic components and the resulting O2 (?-) generation. Cell-free assays consist of a mixture of the individual components of the NADPH oxidase complex, derived from resting phagocytes or in the form of purified recombinant proteins, exposed in vitro to an activating agent (distinct from and unrelated to whole cell stimulants), in the presence of NADPH and oxygen. Activation is commonly quantified by measuring the primary product of the reaction, O2 (?-), trapped immediately after its generation by an appropriate acceptor in a kinetic assay, permitting the calculation of the linear rate of O2 (?-) production, but numerous variations exist, based on the assessment of reaction products or the consumption of substrates. Cell-free assays played a paramount role in the identification and characterization of the components of the NADPH oxidase complex, the deciphering of the mechanisms of assembly, the search for inhibitory drugs, and the diagnosis of various forms of chronic granulomatous disease (CGD). PMID:24504963

Pick, Edgar

2014-01-01

340

Cell Kinase Activity Assay Based on Surface Enhanced Raman Spectroscopy  

PubMed Central

Kinases control many important aspects of cell behavior, such as signal transduction, growth/differentiation, and tumorogenesis. Current methods for assessing kinase activity often require specific antibodies, and/or radioactive labeling. Here we demonstrated a novel detection method to assess kinase activity based on surface enhanced Raman spectroscopy (SERS). Raman signal was obtained after amplification by silver nanoparticles. The sensitivity of this method was comparable to fluorescence measurement of peptide concentration. When purified kinase enzyme was used, the detection limit was comparable to conventional radio-labeling method. We further demonstrated the feasibility to measure kinase activity in crude cell lysate. We suggested this SERS-based kinase activity assay could be a new tool for biomedical research and application.

Yue, Zhicao; Zhuang, Fengfeng; Kumar, Rajar; Wong, Ieong; Cronin, Stephen B; Liu, Yi-Hsin

2009-01-01

341

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

PubMed Central

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

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

2011-01-01

342

Evaluation of the genotoxic potential of the Casearia sylvestris extract on HTC and V79 cells by the comet assay.  

PubMed

Casearia sylvestris is common in tropical America growing wild in Brazil in the states of Amazonas and São Paulo. Its leaves are used in Brazilian folk medicine for several diseases. The present investigation was carried out to examine the genotoxic effects of a C. sylvestris crude ethanolic extract on Hepatoma Tissue Culture (HTC cells) of Rattus norvegicus and Chinese hamster V79 cells in culture, using the comet assay. For the genotoxic evaluation the cells were treated with three different concentrations (0.5, 1 and 2 mg/ml) of extract prepared from a 25 mg/ml aqueous solution. The positive control was cyclophosphamide for HTC cells and methyl methanesulfonate for V79 cells. The duration of the treatment was 2 h. The results showed that the extract of C. sylvestris presented no genotoxic effects and not modified effect inducing DNA damage by alkylating agents cyclophosphamide and methyl methanesulfonate in HTC and V 79 cells respectively. PMID:15046781

Maistro, E L; Carvalho, J C T; Mantovani, M S

2004-06-01

343

Sodium 22+ washout from cultured rat cells  

SciTech Connect

The washout of Na/sup +/ isotopes from tissues and cells is quite complex and not well defined. To further gain insight into this process, we have studied /sup 22/Na/sup +/ washout from cultured Wistar rat skin fibroblasts and vascular smooth muscle cells (VSMCs). In these preparations, /sup 22/Na/sup +/ washout is described by a general three-exponential function. The exponential factor of the fastest component (k1) and the initial exchange rate constant (kie) of cultured fibroblasts decrease in magnitude in response to incubation in K+-deficient medium or in the presence of ouabain and increase in magnitude when the cells are incubated in a Ca++-deficient medium. As the magnitude of the kie declines (in the presence of ouabain) to the level of the exponential factor of the middle component (k2), /sup 22/Na/sup +/ washout is adequately described by a two-exponential function. When the kie is further diminished (in the presence of both ouabain and phloretin) to the range of the exponential factor of the slowest component (k3), the washout of /sup 22/Na/sup +/ is apparently monoexponential. Calculations of the cellular Na/sup +/ concentrations, based on the /sup 22/Na/sup +/ activity in the cells at the initiation of the washout experiments, and the medium specific activity agree with atomic absorption spectrometry measurements of the cellular concentration of this ion. Thus, all three components of /sup 22/Na/sup +/ washout from cultured rat cells are of cellular origin. Using the exponential parameters, compartmental analyses of two models (in parallel and in series) with three cellular Na/sup +/ pools were performed. The results indicate that, independent of the model chosen, the relative size of the largest Na+ pool is 92-93% in fibroblasts and approximately 96% in VSMCs. This pool is most likely to represent the cytosol.

Kino, M.; Nakamura, A.; Hopp, L.; Kuriyama, S.; Aviv, A.

1986-10-01

344

A Rapid and Sensitive Method for Measuring N-Acetylglucosaminidase Activity in Cultured Cells  

PubMed Central

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

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

2013-01-01

345

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

PubMed

A rapid and sensitive method to quantitatively assess N-acetylglucosaminidase (NAG) activity in cultured cells is highly desirable for both basic research and clinical studies. NAG activity is deficient in cells from patients with Mucopolysaccharidosis type IIIB (MPS IIIB) due to mutations in NAGLU, the gene that encodes NAG. Currently available techniques for measuring NAG activity in patient-derived cell lines include chromogenic and fluorogenic assays and provide a biochemical method for the diagnosis of MPS IIIB. However, standard protocols require large amounts of cells, cell disruption by sonication or freeze-thawing, and normalization to the cellular protein content, resulting in an error-prone procedure that is material- and time-consuming and that produces highly variable results. Here we report a new procedure for measuring NAG activity in cultured cells. This procedure is based on the use of the fluorogenic NAG substrate, 4-Methylumbelliferyl-2-acetamido-2-deoxy-alpha-D-glucopyranoside (MUG), in a one-step cell assay that does not require cell disruption or post-assay normalization and that employs a low number of cells in 96-well plate format. We show that the NAG one-step cell assay greatly discriminates between wild-type and MPS IIIB patient-derived fibroblasts, thus providing a rapid method for the detection of deficiencies in NAG activity. We also show that the assay is sensitive to changes in NAG activity due to increases in NAGLU expression achieved by either overexpressing the transcription factor EB (TFEB), a master regulator of lysosomal function, or by inducing TFEB activation chemically. Because of its small format, rapidity, sensitivity and reproducibility, the NAG one-step cell assay is suitable for multiple procedures, including the high-throughput screening of chemical libraries to identify modulators of NAG expression, folding and activity, and the investigation of candidate molecules and constructs for applications in enzyme replacement therapy, gene therapy, and combination therapies. PMID:23840811

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

2013-01-01

346

A new method for the simultaneous analysis of growth and death of immunophenotypically defined cells in culture  

Microsoft Academic Search

Background: Internal standards have been used in flow cytometry methods to enumerate lymphoid subsets and hemopoietic progenitor cells ex vivo. However, the cur- rently available methods cannot be readily applied to the analysis of cultured cells because of the frequent occur- rence of cell death during in vitro assays. Methods: This paper reports a new method for the enu- meration

Alfredo Prieto; Eduardo Reyes; David Diaz; Jorge Monserrat; Esperanza Perucha; Ricardo Vangioni; Antonio de la Hera; Alberto Orfao; Melchor Alvarez-Mon

2000-01-01

347

Validation and implementation of the GeneXpert MRSA/SA blood culture assay in a pediatric setting.  

PubMed

Blood cultures positive for gram-positive cocci in clusters can pose a dilemma for empiric antimicrobial therapy because they could represent coagulase-negative staphylococcus or Staphylococcus aureus bacteremia. The GeneXpert MRSA/SA BC Assay (Cepheid, Sunnyvale, CA) is a polymerase chain reaction-based method for identifying S aureus and methicillin resistance that has been approved for use in adults, but data on its use in samples from pediatric patients is limited. We validated the Xpert MRSA/SA BC Assay for use with anaerobic and polymicrobial specimens from pediatric patients and implemented it for routine presumptive identification of S aureus in our pediatric hospital. The assay was 100% sensitive and specific for methicillin-resistant S aureus and 100% sensitive and 99.5% specific for methicillin-susceptible S aureus. Time to presumptive identification of S aureus bacteremia and determination of methicillin susceptibility was reduced by more than 24 hours. We found the Xpert MRSA/SA BC Assay to be a rapid, accurate tool for detecting methicillin-resistant and methicillin-susceptible S aureus in positive pediatric blood cultures, including polymicrobial cultures and those recovered in anaerobic blood culture media. PMID:22031306

Spencer, David H; Sellenriek, Patricia; Burnham, Carey-Ann D

2011-11-01

348

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

349

CALCIUM OXALATE CRYSTAL ATTACHMENT TO CULTURED KIDNEY EPITHELIAL CELL LINES  

Microsoft Academic Search

PurposeCultured kidney epithelial cell lines have frequently been used in urolithiasis research, and in particular in studies related to the interactions between stone crystals and cell membranes. There is evidence that when epithelial cell lines are transformed or serially passed to immortalize them, they experience changes in both cell physiology and morphology. Stone research utilizing cell cultures is frequently necessary

MICHAEL W. BIGELOW; JOHN H. WIESSNER; JACK G. KLEINMAN; NEIL S. MANDEL

1998-01-01

350

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

351

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

352

A cell culturing system that integrates the cell loading function on a single platform and evaluation of the pulsatile pumping effect on cells.  

PubMed

In this paper, we present a novel microfluidic system with pulsatile cell storing, cell-delivering and cell culturing functions on a single PDMS platform. For this purpose, we have integrated two reservoirs, a pulsatile pumping system containing two soft check valves, which were fabricated by in situ photopolymerization, six switch valves, and three cell culture chambers all developed through a simple and rapid fabrication process. The sample volume delivered per stroke was 120 nl and the transported volume was linearly related to the pumping frequency. We have investigated the effect of the pulsatile pneumatic micropumping on the cells during transport. For this purpose, we pumped two types of cell suspensions, one containing human breast adenocarcinoma cells (MCF-7) and the other mesenchymal stem cells (hMSCs) derived from bone marrow. The effect of pulsatile pumping on both cell types was examined by short and long-term culture experiments. Our results showed that the characteristics of both cells were maintained; they were not damaged by the pumping system. Evaluations were carried out by morphological inspection, viability assay and immunophenotyping analysis. The delivered MCF-7 cells and hMSCs spread and proliferated onto the gelatin coated cell culture chamber. This total micro cell culture system can be applied to cell-based high throughput screening and for co-culture of different cells with different volume. PMID:17624619

Kim, J Y; Park, H; Kwon, K H; Park, J Y; Baek, J Y; Lee, T S; Song, H R; Park, Y D; Lee, S H

2008-02-01

353

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

354

UV inactivation of adenovirus type 41 measured by cell culture mRNA RT-PCR.  

PubMed

Adenoviruses are among the most resistant waterborne pathogens to UV disinfection, yet of the 51 serologically distinct human adenoviruses, only a few have been evaluated for their sensitivities to UV irradiation. Human enteric adenoviruses (Ad40 and Ad41) are difficult to cultivate and reliably assay for infectivity, requiring weeks to obtain cytopathogenic effects (CPE). Inoculated cell cultures often deteriorate before the appearance of distinctive CPE making it difficult to obtain reliable and reproducible data regarding UV inactivation. Adenovirus is a double-stranded DNA virus and produces messenger RNA (mRNA) during replication in host cells. The presence of viral mRNA in host cells is definitive evidence of infection. We recently developed a rapid and reliable cell culture-mRNA RT-PCR assay to detect and quantify adenovirus infectivity. Viral mRNA recovered from cell cultures 5-7 days after infection was purified on oligo-dT latex, treated with DNase, and amplified by RT-PCR using the primers specific for a conserved region of the hexon late mRNA transcript. Treatment of approximately 10(4) Ad41 with different doses of 254 nm germicidal UV radiation resulted in a dose-dependent loss of infectivity. As UV doses were increased from 75 to 200 mJ/cm2, virus survival decreased and no virus infectivity (measured by detectable mRNA) was found at a dose of 225 mJ/cm2 or higher. Our results using the cell culture mRNA RT-PCR assay indicate that Ad41 is more resistant to UV radiation than in a previous study using a conventional cell culture infectivity assay. Results were more similar to those found for Ad 40 using CPE as a measure of infectivity in another previous study. PMID:16046229

Ko, Gwangpyo; Cromeans, Theresa L; Sobsey, Mark D

2005-09-01

355

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

356

Cell damage by UVA radiation of a mercury microscopy lamp 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. Exposure of Chinese hamster ovary cells to ultraviolet-A (UVA) radiation > 10 kJ/m2 resulted in significant modifications of nicotinamide adenine dinucleotide attributed autofluorescence and 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, Tatiana B.; Bauer, Eckhard; Fiedler, Ursula; Berns, Michael W.; Tromberg, Bruce J.; Greulich, Karl O.

1996-04-01

357

Manipulating gene expression and signaling activity in cultured mouse limb bud cells.  

PubMed

Background: The vertebrate limb bud is a well-established system for studying the mechanisms driving growth and patterning of an embryonic tissue. However, approaches for manipulating gene expression are currently limited to time-consuming methods. Culturing primary limb bud cells could potentially be used as a quicker assay. However, limb cells in culture quickly differentiate into cartilage under normal conditions, and approaches delivering DNA and siRNA into primary limb cells in culture are limited. These technical limitations have restricted the utility of limb buds for investigating problems that require higher-throughput approaches. Results: In this report, we describe adaptations to a method for culturing primary limb bud cells in a pre-chondrogenic state, and generate a population of mouse primary limb cells that are responsive to Hedgehog (Hh) signaling. Hh-stimulated cells upregulate Hh target genes as well as an exogenous Hh-responsive reporter. We then describe a method for highly efficient delivery of plasmids and siRNAs into cultured primary limb bud cells in a 96-well format. Conclusions: Cultures of primary limb bud cells are amenable to gene manipulation under conditions that maintain the limb cells in an Hh-responsive, undifferentiated state. This approach provides a medium-throughput system to manipulate gene expression, and test DNA regulatory elements. Developmental Dynamics 243:928-936, 2014. © 2014 Wiley Periodicals, Inc. PMID:24633820

Lewandowski, Jordan P; Pursell, Taylor A; Rabinowitz, Adam H; Vokes, Steven A

2014-07-01

358

Adult mouse hematopoietic stem cells: purification and single-cell assays  

Microsoft Academic Search

Mouse hematopoietic stem cells (HSCs) are the best-studied stem cells because functional assays for mouse HSCs were established earliest and purification techniques for mouse HSCs have progressed furthest. Here we describe our current protocols for the purification of CD34?\\/lowc-Kit+Sca-1+lineage marker? (CD34?KSL) cells, the HSC population making up approximately 0.005% of bone marrow cells in adult C557BL\\/6 mice. Purified HSCs have

Hideo Ema; Yohei Morita; Satoshi Yamazaki; Azusa Matsubara; Jun Seita; Yuko Tadokoro; Hiroyoshi Kondo; Hina Takano; Hiromitsu Nakauchi

2007-01-01

359

Darwinian evolution of prions in cell culture.  

PubMed

Prions are infectious proteins consisting mainly of PrP(Sc), a beta sheet-rich conformer of the normal host protein PrP(C), and occur in different strains. Strain identity is thought to be encoded by PrP(Sc) conformation. We found that biologically cloned prion populations gradually became heterogeneous by accumulating "mutants," and selective pressures resulted in the emergence of different mutants as major constituents of the evolving population. Thus, when transferred from brain to cultured cells, "cell-adapted" prions outcompeted their "brain-adapted" counterparts, and the opposite occurred when prions were returned from cells to brain. Similarly, the inhibitor swainsonine selected for a resistant substrain, whereas, in its absence, the susceptible substrain outgrew its resistant counterpart. Prions, albeit devoid of a nucleic acid genome, are thus subject to mutation and selective amplification. PMID:20044542

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

2010-02-12

360

Isolation and Culture of Larval Cells from C. elegans  

PubMed Central

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 7.8×104 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.

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

2011-01-01

361

Design and Assay of Inhibitors of HIV1 Vpr Cell Killing and Growth Arrest Activity Using Microbial Assay Systems  

Microsoft Academic Search

Viral protein R (Vpr), one of the accessory gene products encoded by the human immunodeficiency virus type 1 (HIV-1) genome, has a number of functions, including causing a growth arrest of HIV-1-infected cells and possibly the death of uninfected bystander cells. In microbial assay systems, the C-terminal portion of Vpr can cause cell death when added externally, and when expressed

Sonia E. Sankovich; Daniela Koleski; Jonathan Baell; Barry Matthews; Ahmed A. Azad; Ian G. Macreadie

1998-01-01

362

Infectious Center Assay of Intracellular Virus and Infective Virus Titer for Equine Mononuclear Cells Infected in vivo and in vitro with Equine Herpesviruses  

PubMed Central

A novel, simple method of infectious center assay was developed to detect and quantitate the intracellular existence of equine herpesvirus 1 and equine herpesvirus 2 in peripheral blood mononuclear cells infected in vivo and in vitro with the viruses by cocultivation of these cells with a permissive equine cell culture. The infectious center titers were correlated with the infectious virus titers. In vivo equine herpesvirus 1-infected mononuclear cells obtained from ponies experimentally infected with the virus and equine herpesvirus 2-infected mononuclear cells obtained from selected naturally infected ponies with the virus gave by infectious center assay a mean value of 67 infectious center/2 x 106 cells as a peak titer on day 4 postinfection and 26 infectious center/2 x 106 cells for equine herpesvirus 1 and equine herpesvirus 2 respectively. The mononuclear cells, in both cases, did not contain detectable infectious virus, but the infectious virus was detected from the respective cells when they were cultured in the presence of mitogen. The equine herpesvirus 1 infected mononuclear cells in culture gave a mean count of 8.05 x 102 infectious center/2 x 106 cells/mL and contained 1.08 x 104 plague assay/mL of infectious virus. Similarly the equine herpesvirus 2 infected mononuclear cells in culture gave a mean count of 7.1 x 101 infectious center/2 x 106 cells/mL and contained <101 tissue culture infective dose50/mL of infectious virus. Mononuclear cells infected in vitro with equine herpesvirus 1 gave a mean count of 9.3 x 104 infectious center/2 x 106 cells/mL and contained 5.75 x 103 plaque assay/mL of infectius virus. Culturing these cells in the presence of mitogen gave a mean count of 5.5 x 103 infectious center/2 x 106 cells/mL and contained 9 x 103 plague assay/mL of infectious virus. A correlation between infectious center assay and infectious virus assay is discussed. ImagesFig. 1.Fig. 2.

Dutta, S.K.; Myrup, A.C.

1983-01-01

363

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

364

Initiation and growth characterization of some hop cell suspension cultures  

Microsoft Academic Search

Cell suspension cultures of some hop (Humulus lupulus L.) cultivars were initiated from corresponding callus cultures induced on different media. Dissimilation curves were determined to characterize the growth of the suspension cultures maintained in Gamborg's B5 and in Murashige and Skoog's medium. Both media proved to be suitable; a comparison of the curves obtained for suspension cultures of the hop

Carlos R. Langezaal; Johannes J. C. Scheffer

1992-01-01

365

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

366

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.

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

367

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.

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

368

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

369

Amino Acid Transport into Cultured Tobacco Cells  

PubMed Central

The effects of calcium ions on lysine transport into cultured Wisconsin-38 tobacco cells were examined. In the presence of calcium, lysine was transported at a relatively low rate for 30 to 40 minutes followed by a period of increasing rates and subsequent stabilization at a higher rate after 2 to 3 hours. In the absence of calcium, transport was uniformly low. Time-dependent stimulation of lysine transport rate was observed after the cells had been preincubated in calcium-containing media. Similar treatments also resulted in the stimulated uptake of a variety of other amino acids, organic compounds, and sulfate. The stimulation of lysine uptake was apparently not due to nutrient starvation. Lysine transport was not stimulated in a time-dependent fashion by K+, La3+, Mg2+, or Mn2+. Cells with stimulated transport rates continued to exhibit high rates when washed with calcium-containing media followed by transport in calcium-containing media. All other cation wash treatments were inhibitory, although magnesium treatments resulted in partial preservation of stimulated transport rates. Cycloheximide inhibited the calcium/time-dependent stimulation of lysine transport and caused the stimulated rate to decay. The initial experimental treatments or the culture conditions may represent some form of shock that alters the membrane transport mechanism, thus reducing transport. The observed calcium/time-dependent stimulation may require protein synthesis and represents damage repair.

Harrington, H. Michael; Berry, Sandra L.; Henke, Randolph R.

1981-01-01

370

Peroxisome dynamics in cultured mammalian cells.  

PubMed

Despite the identification and characterization of various proteins that are essential for peroxisome biogenesis, the origin and the turnover of peroxisomes are still unresolved critical issues. In this study, we used the HaloTag technology as a new approach to examine peroxisome dynamics in cultured mammalian cells. This technology is based on the formation of a covalent bond between the HaloTag protein--a mutated bacterial dehalogenase which is fused to the protein of interest--and a synthetic haloalkane ligand that contains a fluorophore or affinity tag. By using cell-permeable ligands of distinct fluorescence, it is possible to image distinct pools of newly synthesized proteins, generated from a single genetic HaloTag-containing construct, at different wavelengths. Here, we show that peroxisomes display an age-related heterogeneity with respect to their capacity to incorporate newly synthesized proteins. We also demonstrate that these organelles do not exchange their protein content. In addition, we present evidence that the matrix protein content of pre-existing peroxisomes is not evenly distributed over new organelles. Finally, we show that peroxisomes in cultured mammalian cells, under basal growth conditions, have a half-life of approximately 2 days and are mainly degraded by an autophagy-related mechanism. The implications of these findings are discussed. PMID:19719477

Huybrechts, Sofie J; Van Veldhoven, Paul P; Brees, Chantal; Mannaerts, Guy P; Los, Georgyi V; Fransen, Marc

2009-11-01

371

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

PubMed Central

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

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

2012-01-01

372

Assay validation for the assessment of adipogenesis of multipotential stromal cells--a direct comparison of four different methods  

PubMed Central

Background aims Mesenchymal stromal cells (MSCs) are regenerative and immuno-privileged cells that are used for both tissue regeneration and treatment of severe inflammation-related disease. For quality control of manufactured MSC batches in regard to mature fat cell contamination, a quantitative method for measuring adipogenesis is needed. Methods Four previously proposed methods were validated with the use of bone marrow (BM) MSCs during a 21-day in vitro assay. Oil red staining was scored semiquantitatively; peroxisome proliferator activated receptor-? and fatty acid binding protein (FABP)4 transcripts were measured by quantitative real-time polymerase chain reaction; FABP4 protein accumulation was evaluated by flow cytometry; and Nile red/4?,6-diamidino-2-phenylindole (DAPI) ratios were measured in fluorescent microplate assay. Skin fibroblasts and MSCs from fat pad, cartilage and umbilical cord were used as controls. Results Oil red staining indicated considerable heterogeneity between BM donors and individual cells within the same culture. FABP4 transcript levels increased 100- to 5000-fold by day 21, with large donor variability observed. Flow cytometry revealed increasing intra-culture heterogeneity over time; more granular cells accumulated more FABP4 protein and Nile red fluorescence compared with less granular cells. Nile red increase in day-21 MSCs was ?5- and 4-fold, measured by flow cytometry or microplate assay, respectively. MSC proliferation/apoptosis was accounted through the use of Nile red/DAPI ratios; adipogenesis levels in day-21 BM MSCs increased ?13-fold, with significant correlations with oil red scoring observed for MSC from other sources. Conclusions Flow cytometry permits the study of MSC differentiation at the single-cell level and sorting more and less mature cells from mixed cell populations. The microplate assay with the use of the Nile red/DAPI ratio provides rapid quantitative measurements and could be used as a low-cost, high-throughput method to quality-control MSC batches from different tissue sources.

Aldridge, Andrew; Kouroupis, Dimitrios; Churchman, Sarah; English, Anne; Ingham, Eileen; Jones, Elena

2013-01-01

373

A three-dimensional organotypic assay to measure target cell killing by cytotoxic T lymphocytes  

Microsoft Academic Search

Cytotoxic T lymphocytes (CTL) mediate antigen- and cell–cell contact dependent killing of target cells, such as cancer cells and virus-infected cells. In vivo, this process requires the active migration of CTL towards and away from target cells. We here describe an organotypic 3D collagen matrix assay to monitor CTL migration together with CTL-mediated killing of target cells. The assay supports

Bettina Weigelin; Peter Friedl

2010-01-01

374

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

375

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.

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

2014-01-01

376

Real-time quantitative fluorescence measurement of microscale cell culture analog systems  

Microsoft Academic Search

A microscale cell culture analog (muCCA) is a cell-based lab-on-a-chip assay that, as an animal surrogate, is applied to pharmacological studies for toxicology tests. A muCCA typically comprises multiple chambers and microfluidics that connect the chambers, which represent animal organs and blood flow to mimic animal metabolism more realistically. A muCCA is expected to provide a tool for high-throughput drug

Taek-il Oh; Donghyun Kim; Daniel Tatosian; Jong Hwan Sung; Michael Shuler

2007-01-01

377

Identification and Autoregulation of Receptor for OX-LDL in Cultured Human Coronary Artery Endothelial Cells  

Microsoft Academic Search

Although macrophages scavenge oxidatively modified low density lipoprotein (ox-LDL) via specific receptors, the uptake of ox-LDL by endothelial cells is thought to be mediated by a different receptor (LOX-1). We examined the presence of LOX-1 on cultured human coronary artery endothelial cells (HCAECs) by RT-PCR, radioligand blot, and binding assays. LOX-1 mRNA and protein were consistently identified in HCAECs. [125I]-ox-LDL

J. L. Mehta; D. Y. Li

1998-01-01

378

Direct contact cytotoxicity assays for filter-collected, carbonaceous (soot) nanoparticulate material and observations of lung cell response  

NASA Astrophysics Data System (ADS)

A simple, direct contact, cytotoxicity (in vitro) assay has been developed where particulate matter (PM) collected on glass fiber filters was exposed to human epithelial (lung) cells. Carbonaceous (soot) PM included tire, wood, diesel, candle, and variously combusted natural gas PM from a kitchen stove range. Black carbon PM and a commercial multiwall carbon nanotube aggregate PM was also examined in vitro as surrogate materials, and all experimental PM was characterized by field emission scanning electron microscopy and transmission electron microscopy. Assay results for 48 h cultures showed toxicity for all carbonaceous PM with various natural gas PM being the most toxic; this was comparable to the toxicity induced by the surrogate PM. Light microscopy examination of affected epithelial cells confirmed the semi-quantitative results. Comparison of polycyclic aromatic hydrocarbon (PAH) content and concentration for the carbonaceous PM showed no PAH correlation with relative cell viability (cell death) after 48 h.

Soto, K. F.; Garza, K. M.; Shi, Y.; Murr, L. E.

379

A protocol for in situ enzyme assays to assess the differentiation of human intestinal Caco-2 cells.  

PubMed

The Caco-2 cell line spontaneously differentiates into polarised enterocytes expressing high levels of brush border enzymes typical of small intestinal epithelial cells (peptidases, alkaline phosphatase, disaccharidases). The activities of these enzymes gradually increase after cell confluence reaching a plateau after 2-3 weeks of culture and can be used as reliable markers to evaluate differentiation of Caco-2 cells. We have developed a rapid in situ method on live cells to measure activities of alkaline phosphatase, alanyl amino peptidase and sucrase. The substrates were added to the apical compartment of confluent cells maintained for 8, 15 and 21 days on polycarbonate filter inserts and sampling was performed at time intervals. Alkaline phosphatase and alanyl aminopeptidase were assayed using as substrates p-Nitrophenyl phosphate and alanine-p-nitroanilide, respectively, and the yellow product detected spectrophotometrically at 405 nm. Sucrase activity was measured as the release of glucose from sucrose using a fluorimetric assay (Amplex® Red Glucose Assay Kit) in which H(2)O(2), produced by the coupled glucose oxidase/horseradish peroxidase reactions, oxidises the colourless reagent to red-fluorescent resorufin. All these assays are rapid and reproducible and can easily be adapted to robotised high throughput platforms. PMID:22123491

Ferruzza, Simonetta; Rossi, Carlotta; Scarino, Maria Laura; Sambuy, Yula

2012-12-01

380

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