WorldWideScience

Sample records for cell labeling research

  1. Clinical applications of cells labelling

    International Nuclear Information System (INIS)

    Blood cells labelled with radionuclides are reviewed and main applications are described. Red blood cell labelling by both random and specific principle. A table with most important clinical uses, 99mTc labelling of RBC are described pre tinning and in vivo reduction of Tc, in vitro labelling and administration of labelled RBC and in vivo modified technique. Labelled leucocytes with several 99mTc-complex radiopharmaceuticals by in vitro technique and specific monoclonal s for white cells(neutrofiles). Labelled platelets for clinical use and research by in vitro technique and in vivo labelling

  2. Tritium labeling for bio-med research

    International Nuclear Information System (INIS)

    A very large fraction of what we know about biochemical pathways in the living cell has resulted from the use of radioactively-labeled tracer compounds; the use of tritium-labeled compounds has been particularly important. As research in biochemistry and biology has progressed the need has arisen to label compounds of higher specific activity and of increasing molecular complexity - for example, oligo-nucleotides, polypeptides, hormones, enzymes. Our laboratory has gradually developed special facilities for handling tritium at the kilocurie level. These facilities have already proven extremely valuable in producing labeled compounds that are not available from commercial sources. The principal ways employed for compound labeling are: (1) microwave discharge labeling, (2) catalytic tritio-hydrogenation, (3) catalytic exchange with T2O, and (4) replacement of halogen atoms by T. Studies have also been carried out on tritiation by the replacement of halogen atoms with T atoms. These results indicate that carrier-free tritium-labeled products, including biomacromolecules, can be produced in this way

  3. Label-Free Biosensors for Cell Biology

    Directory of Open Access Journals (Sweden)

    Ye Fang

    2011-01-01

    Full Text Available Label-free biosensors for studying cell biology have finally come of age. Recent developments have advanced the biosensors from low throughput and high maintenance research tools to high throughput and low maintenance screening platforms. In parallel, the biosensors have evolved from an analytical tool solely for molecular interaction analysis to powerful platforms for studying cell biology at the whole cell level. This paper presents historical development, detection principles, and applications in cell biology of label-free biosensors. Future perspectives are also discussed.

  4. A brief history of cell labelling

    Energy Technology Data Exchange (ETDEWEB)

    Peters, A.M. [Royal Sussex Country Hospital, Brighton (United Kingdom)

    2005-12-15

    The term cell labelling is usually used in the context of labelled leukocytes for imaging inflammation and labelled platelets for imaging thrombosis. Erythrocyte labelling for in vitro measurements of red cell life span, in vivo measurements of splenic red cell pooling, radionuclide ventriculography and imaging sites of bleeding has developed rather separately and has a different history. Labelled platelets and leukocytes were originally developed for cell kinetic studies. Since the current-day applications of labelled platelets and leukocytes depend on a clear understanding of cell kinetics, these classical studies are important and relevant to the history of cell labelling.

  5. Label-Free Biosensors for Cell Biology

    OpenAIRE

    Ye Fang

    2011-01-01

    Label-free biosensors for studying cell biology have finally come of age. Recent developments have advanced the biosensors from low throughput and high maintenance research tools to high throughput and low maintenance screening platforms. In parallel, the biosensors have evolved from an analytical tool solely for molecular interaction analysis to powerful platforms for studying cell biology at the whole cell level. This paper presents historical development, detection principles, and applicat...

  6. Research of private label development in Croatia

    Directory of Open Access Journals (Sweden)

    Sandra Horvat

    2009-07-01

    Full Text Available Private labels have been present on the market since 19th century but their intensive market growth began in the last thirty years after retailers realized what their potential could be in the fight against ever-growing competition. Their market growth has not been distributed equally thought the world so Europe became the region with the highest private label market share, which exceeds 40% on some markets. Although the private label market share in Croatia is considerably smaller, it has also increased steadily over the last decade since private labels were introduced on the market. This paper presents the findings of a research conducted for the purpose of identifying trends in private label development on the Croatian market. The research was conducted through in-depth interviews with private label managers in retail companies in Croatia, and with the managers responsible for private label production in manufacturing companies. The research identified three expected trends of private label development in Croatia and these are: an increase in private label quality, the maintenance of a price gap between private labels and manufacturers’ brands and a further increase in the private label market share.

  7. The antibody approach of labeling blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.

    1992-12-31

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are critically assessed and evaluated.

  8. The antibody approach of labeling blood cells

    International Nuclear Information System (INIS)

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are critically assessed and evaluated

  9. The antibody approach of labeling blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.

    1991-01-01

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

  10. The antibody approach of labeling blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.

    1991-12-31

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

  11. Research with Individuals Labeled "Other": Reflections on the Research Process

    Science.gov (United States)

    Petersen, Amy J.

    2011-01-01

    Using the emancipatory research paradigm as a conceptual framework, this autoethnography reflects upon participant and researcher relationships within a larger qualitative research study that involved participants labeled "other". Issues relating to fear of the "other", building reciprocal relationships, and who gains from the research are…

  12. Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy

    Science.gov (United States)

    Kemper, Björn; Bauwens, Andreas; Vollmer, Angelika; Ketelhut, Steffi; Langehanenberg, Patrik; Müthing, Johannes; Karch, Helge; von Bally, Gert

    2010-05-01

    Digital holographic microscopy (DHM) enables quantitative multifocus phase contrast imaging for nondestructive technical inspection and live cell analysis. Time-lapse investigations on human brain microvascular endothelial cells demonstrate the use of DHM for label-free dynamic quantitative monitoring of cell division of mother cells into daughter cells. Cytokinetic DHM analysis provides future applications in toxicology and cancer research.

  13. Stable isotope labeling of oligosaccharide cell surface antigens

    Energy Technology Data Exchange (ETDEWEB)

    Unkefer, C.J.; Silks, L.A. III; Martinez, R.A. [and others

    1998-12-31

    The overall goal of this Laboratory Directed Research and Development (LDRD) project was to develop new methods for synthesis of {sup 13}C-labeled oligosaccharides that are required for nuclear magnetic resonance (NMR) studies of their solution conformation. Oligosaccharides are components of the cell`s outer surface and are involved in important processes such as cell-cell recognition and adhesion. Recently, Danishefsky and coworkers at Slone-Kettering Cancer Center developed a method for the solid-phase chemical synthesis of oligosaccharides. The specific goal of this LDRD project was to prepare uniform {sup 13}C-labeled aldohexose precursors required for the solid-phase synthesis of the Lewis blood-group antigenic determinants. We report the synthesis of {sup 13}C-labeled D-glucal, D-galactal and Fucosyl precursors. We have been collaborating with the Danishefsky group on the synthesis of the Lewis oligosaccharides and the NMR analysis of their solution conformation.

  14. Aptamer-mediated indirect quantum dot labeling and fluorescent imaging of target proteins in living cells

    International Nuclear Information System (INIS)

    Protein labeling for dynamic living cell imaging plays a significant role in basic biological research, as well as in clinical diagnostics and therapeutics. We have developed a novel strategy in which the dynamic visualization of proteins within living cells is achieved by using aptamers as mediators for indirect protein labeling of quantum dots (QDs). With this strategy, the target protein angiogenin was successfully labeled with fluorescent QDs in a minor intactness model, which was mediated by the aptamer AL6-B. Subsequent living cell imaging analyses indicated that the QDs nanoprobes were selectively bound to human umbilical vein endothelial cells, gradually internalized into the cytoplasm, and mostly localized in the lysosome organelle, indicating that the labeled protein retained high activity. Compared with traditional direct protein labeling methods, the proposed aptamer-mediated strategy is simple, inexpensive, and provides a highly selective, stable, and intact labeling platform that has shown great promise for future biomedical labeling and intracellular protein dynamic analyses. (paper)

  15. A Chemical Probe that Labels Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Nao Hirata

    2014-03-01

    Full Text Available A small-molecule fluorescent probe specific for human pluripotent stem cells would serve as a useful tool for basic cell biology research and stem cell therapy. Screening of fluorescent chemical libraries with human induced pluripotent stem cells (iPSCs and subsequent evaluation of hit molecules identified a fluorescent compound (Kyoto probe 1 [KP-1] that selectively labels human pluripotent stem cells. Our analyses indicated that the selectivity results primarily from a distinct expression pattern of ABC transporters in human pluripotent stem cells and from the transporter selectivity of KP-1. Expression of ABCB1 (MDR1 and ABCG2 (BCRP, both of which cause the efflux of KP-1, is repressed in human pluripotent stem cells. Although KP-1, like other pluripotent markers, is not absolutely specific for pluripotent stem cells, the identified chemical probe may be used in conjunction with other reagents.

  16. Carboxyfluorescein Diacetate Succinimidyl Ester Fluorescent Dye for Cell Labeling

    Institute of Scientific and Technical Information of China (English)

    Xiao-Qi WANG; Xiu-Mei DUAN; Li-Hua LIU; Yan-Qiu FANG; Yan TAN

    2005-01-01

    Our objective was to study the properties of the carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and the methodology of cell labeling using CFDA-SE fluorescent dye. First, we analyzed the kinetics of CFDA-SE fluorescent dye intensity over time. Second, we determined the optimal concentration of CFDA-SE fluorescent dye for cell labeling. Third, we tested the toxicity of CFDA-SE fluorescent dye on labeled cells. Finally, we determined the optimal staining time of CFDA-SE fluorescent dye for cell labeling.The results show that the optimal concentration of CFDA-SE fluorescent dye for cell labeling varies according to different cell types. CFDA-SE fluorescent dye is non-toxic to cells as the cell death rate caused by CFDASE labeling is below 5%. The optimal cell labeling time was determined to be 8 min of incubation with CFDA-SE fluorescent dye. We concluded that the advantages of using CFDA-SE fluorescent dye for cell labeling are as follows: (1) the binding of CFDA-SE fluorescent dye to cells is stable; (2) CFDA-SE fluorescent dye is not toxic and does not modify the viability of labeled cells; and (3) CFDA-SE fluorescent dye is a suitable fluorochrome for cell labeling.

  17. Research on consumer reactions to nutrition labelling (FLABEL)

    DEFF Research Database (Denmark)

    Grunert, Klaus G.

    Nutrition labels are potentially a major instrument for enabling consumers to make healthier food choices, but current insights into how nutrition labels are used by consumers in real-world shopping situations are limited, making the science-based formulation of new labelling policies......' attention-getting potential, on the way consumers draw inferences on product healthiness from them, and on how they actually affect choices. Based on the findings from this project, best practice guidelines will be developed for use of nutrition labelling in EU policy and the food industry, especially SMEs...... and the evaluation of existing ones difficult. Recent and ongoing research, including research in the European Union (EU)-funded FP7 project FLABEL (Food Labelling to Advance Better Education for Life), is accumulating evidence not only on consumer liking of labels and on self-reported use, but also on labels...

  18. Immunogold labels: cell-surface markers in atomic force microscopy

    NARCIS (Netherlands)

    Putman, Constant A.J.; Grooth, de Bart G.; Hansma, Paul K.; Hulst, van Niek F.; Greve, Jan

    1993-01-01

    The feasibility of using immunogold labels as cell-surface markers in atomic force microscopy is shown in this paper. The atomic force microscope (AFM) was used to image the surface of immunogold-labeled human lymphocytes. The lymphocytes were isolated from whole blood and labeled by an indirect imm

  19. Labeling of mesenchymal stem cells by bioconjugated quantum dots.

    Science.gov (United States)

    Shah, Bhranti S; Clark, Paul A; Moioli, Eduardo K; Stroscio, Michael A; Mao, Jeremy J

    2007-10-01

    Long-term labeling of stem cells during self-replication and differentiation benefits investigations of development and tissue regeneration. We report the labeling of human mesenchymal stem cells (hMSCs) with RGD-conjugated quantum dots (QDs) during self-replication, and multilineage differentiations into osteogenic, chondrogenic, and adipogenic cells. QD-labeled hMSCs remained viable as unlabeled hMSCs from the same subpopulation. These findings suggest the use of bioconjugated QDs as an effective probe for long-term labeling of stem cells.

  20. Nanodiamonds with silicon vacancy defects for non-toxic photostable fluorescent labeling of neural precursor cells

    CERN Document Server

    Merson, Tobias D; Aharonovich, Igor; Turbic, Alisa; Kilpatrick, Trevor J; Turnley, Ann M

    2013-01-01

    Nanodiamonds (NDs) containing silicon vacancy (SiV) defects were evaluated as a potential biomarker for the labeling and fluorescent imaging of neural precursor cells (NPCs). SiV-containing NDs were synthesized using chemical vapor deposition and silicon ion implantation. Spectrally, SiV-containing NDs exhibited extremely stable fluorescence and narrow bandwidth emission with an excellent signal to noise ratio exceeding that of NDs containing nitrogen-vacancy (NV) centers. NPCs labeled with NDs exhibited normal cell viability and proliferative properties consistent with biocompatibility. We conclude that SiVcontaining NDs are a promising biomedical research tool for cellular labeling and optical imaging in stem cell research.

  1. Traceless affinity labeling of endogenous proteins for functional analysis in living cells.

    Science.gov (United States)

    Hayashi, Takahiro; Hamachi, Itaru

    2012-09-18

    Protein labeling and imaging techniques have provided tremendous opportunities to study the structure, function, dynamics, and localization of individual proteins in the complex environment of living cells. Molecular biology-based approaches, such as GFP-fusion tags and monoclonal antibodies, have served as important tools for the visualization of individual proteins in cells. Although these techniques continue to be valuable for live cell imaging, they have a number of limitations that have only been addressed by recent progress in chemistry-based approaches. These chemical approaches benefit greatly from the smaller probe sizes that should result in fewer perturbations to proteins and to biological systems as a whole. Despite the research in this area, so far none of these labeling techniques permit labeling and imaging of selected endogenous proteins in living cells. Researchers have widely used affinity labeling, in which the protein of interest is labeled by a reactive group attached to a ligand, to identify and characterize proteins. Since the first report of affinity labeling in the early 1960s, efforts to fine-tune the chemical structures of both the reactive group and ligand have led to protein labeling with excellent target selectivity in the whole proteome of living cells. Although the chemical probes used for affinity labeling generally inactivate target proteins, this strategy holds promise as a valuable tool for the labeling and imaging of endogenous proteins in living cells and by extension in living animals. In this Account, we summarize traceless affinity labeling, a technique explored mainly in our laboratory. In our overview of the different labeling techniques, we emphasize the challenge of designing chemical probes that allow for dissociation of the affinity module (often a ligand) after the labeling reaction so that the labeled protein retains its native function. This feature distinguishes the traceless labeling approach from the traditional

  2. Immunogold labels: cell-surface markers in atomic force microscopy

    OpenAIRE

    Putman, Constant A.J.; Grooth, de, B.G.; Hansma, Paul K.; Hulst, van der, R.W.M.; Greve, Jan

    1993-01-01

    The feasibility of using immunogold labels as cell-surface markers in atomic force microscopy is shown in this paper. The atomic force microscope (AFM) was used to image the surface of immunogold-labeled human lymphocytes. The lymphocytes were isolated from whole blood and labeled by an indirect immunolabeling method using the monoclonal antibody anti-CD3 and a secondary antibody (Goat-anti-Mouse) linked to 30 nm colloidal gold particles. Some of the samples were enhanced by silver deposition...

  3. Instant magnetic labeling of tumor cells by ultrasound in vitro

    International Nuclear Information System (INIS)

    Magnetic labeling of living cells creates opportunities for numerous biomedical applications. Here we describe an instantly cell magnetic labeling method based on ultrasound. We present a detailed study on the ultrasound performance of a simple and efficient labeling protocol for H-22 cells in vitro. High frequency focus ultrasound was investigated as an alternative method to achieve instant cell labeling with the magnetic particles without the need for adjunct agents or initiating cell cultures. Mean diameter of 168 nm dextran-T40 coated superparamagnetic iron oxide (SPIO) nanoparticles were prepared by means of classical coprecipitation in solution in our laboratory. H-22 tumor cells suspended in phosphate-buffered saline (PBS, pH=7.2) were exposed to ultrasound at 1.37 MHz for up to 120 s in the presence of SPIOs. The cellular uptake of iron oxide nanoparticles was detected by prussion blue staining. The viability of cells was determined by a trypan blue exclusion test. At 2 W power and 60 s ultrasound exposure in presence of 410 μg/ml SPIOs, H-22 cell labeling efficiency reached 69.4±6.3% and the labeled cells exhibited an iron content of 10.38±2.43 pg per cell. Furthermore, 95.2±3.2% cells remained viable. The results indicated that the ultrasound protocol could be potentially applied to label cells with large-sized magnetic particles. We also calculated the shear stress at the 2 W power and 1.37 MHz used in experiments. The results showed that the shear stress threshold for ultrasonically induced H-22 cell reparable sonoporation was 697 Pa. These findings provide a quantitative guidance in designing ultrasound protocols for cell labeling. - Highlights: → High frequency focus ultrasound can be used as a safe method for instant magnetic labeling of cells. → 8-16 times increased efficiency can be gained by ultrasound versus that by transfection agents. → Calculation of shear stress around cells provide a quantitative design for ultrasound protocols.

  4. Labeling of lectin receptors during the cell cycle.

    Science.gov (United States)

    Garrido, J

    1976-12-01

    Labeling of lectin receptors during the cell cycle. (Localizabión de receptores para lectinas durante el ciclo celular). Arch. Biol. Med. Exper. 10: 100-104, 1976. The topographic distribution of specific cell surface receptors for concanavalin A and wheat germ agglutinin was studied by ultrastructural labeling in the course of the cell cycle. C12TSV5 cells were synchronized by double thymidine block or mechanical selection (shakeoff). They were labeled by means of lectin-peroxidase techniques while in G1 S, G2 and M phases of the cycle. The results obtained were similar for both lectins employed. Interphase cells (G1 S, G2) present a stlihtly discontinous labeling pattern that is similar to the one observed on unsynchronized cells of the same line. Cells in mitosis, on the contrary, present a highly discontinous distribution of reaction product. This pattern disappears after the cells enters G1 and is not present on mitotic cells fixed in aldehyde prior to labeling. PMID:1030938

  5. Polyelectrolyte coating of ferumoxytol nanoparticles for labeling of dendritic cells

    Science.gov (United States)

    Celikkin, Nehar; Jakubcová, Lucie; Zenke, Martin; Hoss, Mareike; Wong, John Erik; Hieronymus, Thomas

    2015-04-01

    Engineered magnetic nanoparticles (MNPs) are emerging to be used as cell tracers, drug delivery vehicles, and contrast agents for magnetic resonance imaging (MRI) for enhanced theragnostic applications in biomedicine. In vitro labeling of target cell populations with MNPs and their implantation into animal models and patients shows promising outcomes in monitoring successful cell engraftment, differentiation and migration by using MRI. Dendritic cells (DCs) are professional antigen-presenting cells that initiate adaptive immune responses. Thus, DCs have been the focus of cellular immunotherapy and are increasingly applied in clinical trials. Here, we addressed the coating of different polyelectrolytes (PE) around ferumoxytol particles using the layer-by-layer technique. The impact of PE-coated ferumoxytol particles for labeling of DCs and Flt3+ DC progenitors was then investigated. The results from our studies revealed that PE-coated ferumoxytol particles can be readily employed for labeling of DC and DC progenitors and thus are potentially suitable as contrast agents for MRI tracking.

  6. Polyelectrolyte coating of ferumoxytol nanoparticles for labeling of dendritic cells

    International Nuclear Information System (INIS)

    Engineered magnetic nanoparticles (MNPs) are emerging to be used as cell tracers, drug delivery vehicles, and contrast agents for magnetic resonance imaging (MRI) for enhanced theragnostic applications in biomedicine. In vitro labeling of target cell populations with MNPs and their implantation into animal models and patients shows promising outcomes in monitoring successful cell engraftment, differentiation and migration by using MRI. Dendritic cells (DCs) are professional antigen-presenting cells that initiate adaptive immune responses. Thus, DCs have been the focus of cellular immunotherapy and are increasingly applied in clinical trials. Here, we addressed the coating of different polyelectrolytes (PE) around ferumoxytol particles using the layer-by-layer technique. The impact of PE-coated ferumoxytol particles for labeling of DCs and Flt3+ DC progenitors was then investigated. The results from our studies revealed that PE-coated ferumoxytol particles can be readily employed for labeling of DC and DC progenitors and thus are potentially suitable as contrast agents for MRI tracking

  7. Production of Alexa Fluor 488-labeled reovirus and characterization of target cell binding, competence, and immunogenicity of labeled virions.

    Science.gov (United States)

    Fecek, Ronald J; Busch, Ryan; Lin, Hong; Pal, Kasturi; Cunningham, Cynthia A; Cuff, Christopher F

    2006-07-31

    Respiratory enteric orphan virus (reovirus) has been used to study many aspects of the biology and genetics of viruses, viral infection, pathogenesis, and the immune response to virus infection. This report describes the functional activity of virus labeled with Alexa Fluor 488, a stable fluorescent dye. Matrix assisted laser desorption-time of flight analysis indicated that Alexa Fluor 488 labeled the outer capsid proteins of reovirus. Labeled virus bound to murine L929 fibroblasts as determined by flow cytometry and fluorescence microscopy, and the specificity of binding were demonstrated by competitive inhibition with non-labeled virus. Labeled reovirus induced apoptosis and cytopathic effect in infected L929 cells. Mice infected with labeled virus mounted robust serum antibody and CD8(+) T-cell responses, indicating that labeled virus retained immunogenicity in vivo. These results indicate that Alexa Fluor 488-labeled virus provides a powerful new tool to analyze reovirus infection in vitro and in vivo.

  8. A microfluidics-based technique for automated and rapid labeling of cells for flow cytometry

    Science.gov (United States)

    Patibandla, Phani K.; Estrada, Rosendo; Kannan, Manasaa; Sethu, Palaniappan

    2014-03-01

    Flow cytometry is a powerful technique capable of simultaneous multi-parametric analysis of heterogeneous cell populations for research and clinical applications. In recent years, the flow cytometer has been miniaturized and made portable for application in clinical- and resource-limited settings. The sample preparation procedure, i.e. labeling of cells with antibodies conjugated to fluorescent labels, is a time consuming (˜45 min) and labor-intensive procedure. Microfluidics provides enabling technologies to accomplish rapid and automated sample preparation. Using an integrated microfluidic device consisting of a labeling and washing module, we demonstrate a new protocol that can eliminate sample handling and accomplish sample and reagent metering, high-efficiency mixing, labeling and washing in rapid automated fashion. The labeling module consists of a long microfluidic channel with an integrated chaotic mixer. Samples and reagents are precisely metered into this device to accomplish rapid and high-efficiency mixing. The mixed sample and reagents are collected in a holding syringe and held for up to 8 min following which the mixture is introduced into an inertial washing module to obtain ‘analysis-ready’ samples. The washing module consists of a high aspect ratio channel capable of focusing cells to equilibrium positions close to the channel walls. By introducing the cells and labeling reagents in a narrow stream at the center of the channel flanked on both sides by a wash buffer, the elution of cells into the wash buffer away from the free unbound antibodies is accomplished. After initial calibration experiments to determine appropriate ‘holding time’ to allow antibody binding, both modules were used in conjunction to label MOLT-3 cells (T lymphoblast cell line) with three different antibodies simultaneously. Results confirm no significant difference in mean fluorescence intensity values for all three antibodies labels (p < 0.01) between the

  9. CELL RESEARCH

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    REVIEWSInducible resistance to Fas-mediated apoptosis in B cells…………………………………ROTHSTEIN Thomas L (245)Executionary pathway for apoptosis: lessons from mutant mice………………………………………WOO Minna, Razqallah Hakem, Tak W Mak (267)The SHP-2 tyrosine phosphatase: Signaling mechanisms and biological functions…………………………………QU Cheng Kui (279)REGULAR ARTICLESTemperature dependent expression of cdc2 and cyclin B1 in spermatogenic cells during spermatogenesis…………………………KONG Wei Hua, Zheng GU, Jining LU, Jiake TSO (289)Transgenic mice overexpressing γ-aminobutyric acid transporter subtype I develop obesity…………………………………MA Ying Hua, Jia Hua HU, Xiao Gang ZHOU, Ruo Wang ZENG, Zhen Tong MEI, Jian FEI, Li He GUO (303)Genetic aberration in primary hepatocellular carcinoma: correlation between p53 gene mutation and loss-of-heterozygosity on chromosome 16q21-q23 and 9p21-p23………………………………………WANG Gang, Chang Hui HUANG, Yan ZHAO, Ling CAI, Ying WANG, Shi Jin XIU, Zheng Wen JIANG, Shuang YANG, Xin Tai ZHAO, Wei HUANG, Jian Ren GU (311)Identification and genetic mapping of four novel genes that regulate leaf deve- lopment in Arabidopsis………………………………………………SUN Yue, Wei ZHANG, Feng Ling LI, Ying Li GUO, Tian Lei LIU, Hai HUANG (325)NOTICE FOR CONTRIBUTORS…………………………………(337)CONTENTS of Vol. 10, 2000…………………………………………………(338)

  10. Interfacial polymerization for colorimetric labeling of protein expression in cells.

    Directory of Open Access Journals (Sweden)

    Jacob L Lilly

    Full Text Available Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium.

  11. Interfacial polymerization for colorimetric labeling of protein expression in cells.

    Science.gov (United States)

    Lilly, Jacob L; Sheldon, Phillip R; Hoversten, Liv J; Romero, Gabriela; Balasubramaniam, Vivek; Berron, Brad J

    2014-01-01

    Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium.

  12. Deep Learning in Label-free Cell Classification.

    Science.gov (United States)

    Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram

    2016-03-15

    Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individual cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. This system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.

  13. Deep Learning in Label-free Cell Classification

    Science.gov (United States)

    Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K.; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram

    2016-03-01

    Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individual cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. This system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.

  14. Labeling What Some Researchers Are Already Doing: Design Research Updated

    Science.gov (United States)

    Norris, Cathie; Soloway, Elliot; Tan, Chun Ming; Looi, Chee-Kit; Wong, Lung Hsiang

    2013-01-01

    Increasingly, "research" is not being conducted in a separate group in an organization; rather, it is being integrated into the fabric of the organization. Google recently described such an integrated, "hybrid" model of software research that is characterized by cycles of short duration and small-scope activity that is ongoing…

  15. Detection of pulmonary hemorrhage with technetium-labeled red cells

    Energy Technology Data Exchange (ETDEWEB)

    Winzelberg, G.G.; Laman, D.; Sachs, M.; Miller, W.H.

    1981-10-01

    Noninvasive techniques to aid in the diagnosis of massive pulmonary hemoptysis would be helpful in guiding more-invasive procedures such as bronchial artery angiography, which carries a risk of transverse myelitis. A patient was studied with technetium-labeled red cells and successfully detected a site of intermittent hemorrhage from the lung.

  16. Detection of pulmonary hemorrhage with technetium-labeled red cells

    International Nuclear Information System (INIS)

    Noninvasive techniques to aid in the diagnosis of massive pulmonary hemoptysis would be helpful in guiding more-invasive procedures such as bronchial artery angiography, which carries a risk of transverse myelitis. A patient was studied with technetium-labeled red cells and successfully detected a site of intermittent hemorrhage from the lung

  17. Labelling of live cells using fluorescent aptamers: binding reversal with DNA nucleases

    OpenAIRE

    Terazono Hideyuki; Anzai Yu; Soloviev Mikhail; Yasuda Kenji

    2010-01-01

    Abstract A reversible cell labelling method has been developed for non-destructive and non-invasive cell labelling and purification. Our method uses high affinity single strand DNA (ssDNA) aptamers against surface exposed target molecules on cells. The aptamers are subsequently removed from the cell surface using DNase nuclease treatment. We exemplified our method by labelling human acute lymphoblastic leukemia cells with Qdot-ssDNA aptamers, and restoring them to the label-free condition by ...

  18. Polyelectrolyte coating of ferumoxytol nanoparticles for labeling of dendritic cells

    Energy Technology Data Exchange (ETDEWEB)

    Celikkin, Nehar; Jakubcová, Lucie; Zenke, Martin [Institute for Biomedical Engineering, Department of Cell Biology, RWTH Aachen University Hospital, Pauwelsstrasse 30, 52074 Aachen (Germany); Helmholtz Institute for Biomedical Engineering, RWTH Aachen University, Pauwelsstrasse 20, 52074 Aachen (Germany); Hoss, Mareike [Institute of Pathology, Electron Microscopy Facility, RWTH Aachen University Hospital, Pauwelsstrasse 30, 52074 Aachen (Germany); Wong, John Erik, E-mail: John.Wong@avt.rwth-aachen.de [Chemical Process Engineering, RWTH Aachen University, Turmstrasse 46, 52056 Aachen (Germany); DWI – Leibniz Institute for Interactive Materials Research, Forckenbeckstrasse 50, Aachen (Germany); Hieronymus, Thomas, E-mail: thomas.hieronymus@rwth-aachen.de [Institute for Biomedical Engineering, Department of Cell Biology, RWTH Aachen University Hospital, Pauwelsstrasse 30, 52074 Aachen (Germany); Helmholtz Institute for Biomedical Engineering, RWTH Aachen University, Pauwelsstrasse 20, 52074 Aachen (Germany)

    2015-04-15

    Engineered magnetic nanoparticles (MNPs) are emerging to be used as cell tracers, drug delivery vehicles, and contrast agents for magnetic resonance imaging (MRI) for enhanced theragnostic applications in biomedicine. In vitro labeling of target cell populations with MNPs and their implantation into animal models and patients shows promising outcomes in monitoring successful cell engraftment, differentiation and migration by using MRI. Dendritic cells (DCs) are professional antigen-presenting cells that initiate adaptive immune responses. Thus, DCs have been the focus of cellular immunotherapy and are increasingly applied in clinical trials. Here, we addressed the coating of different polyelectrolytes (PE) around ferumoxytol particles using the layer-by-layer technique. The impact of PE-coated ferumoxytol particles for labeling of DCs and Flt3{sup +} DC progenitors was then investigated. The results from our studies revealed that PE-coated ferumoxytol particles can be readily employed for labeling of DC and DC progenitors and thus are potentially suitable as contrast agents for MRI tracking.

  19. Labeling of mesenchymal stem cells for MRI with single-cell sensitivity

    Directory of Open Access Journals (Sweden)

    Ariza de Schellenberger A

    2016-04-01

    Full Text Available Angela Ariza de Schellenberger,1 Harald Kratz,1 Tracy D Farr,2,3 Norbert Löwa,4 Ralf Hauptmann,1 Susanne Wagner,1 Matthias Taupitz,1 Jörg Schnorr,1 Eyk A Schellenberger1 1Department of Radiology, 2Department of Experimental Neurology, Center for Stroke Research Berlin, Charité – Universitätsmedizin Berlin, Berlin, Germany; 3School of Life Sciences, University of Nottingham, Medical School, Nottingham, UK; 4Department of Biomagnetic Signals, Physikalisch-Technische Bundesanstalt Berlin, Berlin, Germany Abstract: Sensitive cell detection by magnetic resonance imaging (MRI is an important tool for the development of cell therapies. However, clinically approved contrast agents that allow single-cell detection are currently not available. Therefore, we compared very small iron oxide nanoparticles (VSOP and new multicore carboxymethyl dextran-coated iron oxide nanoparticles (multicore particles, MCP designed by our department for magnetic particle imaging (MPI with discontinued Resovist® regarding their suitability for detection of single mesenchymal stem cells (MSC by MRI. We achieved an average intracellular nanoparticle (NP load of >10 pg Fe per cell without the use of transfection agents. NP loading did not lead to significantly different results in proliferation, colony formation, and multilineage in vitro differentiation assays in comparison to controls. MRI allowed single-cell detection using VSOP, MCP, and Resovist® in conjunction with high-resolution T2*-weighted imaging at 7 T with postprocessing of phase images in agarose cell phantoms and in vivo after delivery of 2,000 NP-labeled MSC into mouse brains via the left carotid artery. With optimized labeling conditions, a detection rate of ~45% was achieved; however, the experiments were limited by nonhomogeneous NP loading of the MSC population. Attempts should be made to achieve better cell separation for homogeneous NP loading and to thus improve NP

  20. Small Molecule-Photoactive Yellow Protein Labeling Technology in Live Cell Imaging

    Directory of Open Access Journals (Sweden)

    Feng Gao

    2016-08-01

    Full Text Available Characterization of the chemical environment, movement, trafficking and interactions of proteins in live cells is essential to understanding their functions. Labeling protein with functional molecules is a widely used approach in protein research to elucidate the protein location and functions both in vitro and in live cells or in vivo. A peptide or a protein tag fused to the protein of interest and provides the opportunities for an attachment of small molecule probes or other fluorophore to image the dynamics of protein localization. Here we reviewed the recent development of no-wash small molecular probes for photoactive yellow protein (PYP-tag, by the means of utilizing a quenching mechanism based on the intramolecular interactions, or an environmental-sensitive fluorophore. Several fluorogenic probes have been developed, with fast labeling kinetics and cell permeability. This technology allows quick live-cell imaging of cell-surface and intracellular proteins without a wash-out procedure.

  1. Adeno associated viral-mediated intraosseous labeling of bone marrow derived cells for CNS tracking.

    Science.gov (United States)

    Selenica, Maj-Linda B; Reid, Patrick; Pena, Gabriela; Alvarez, Jennifer; Hunt, Jerry B; Nash, Kevin R; Morgan, Dave; Gordon, Marcia N; Lee, Daniel C

    2016-05-01

    Inflammation, including microglial activation in the CNS, is an important hallmark in many neurodegenerative diseases. Microglial stimuli not only impact the brain microenvironment by production and release of cytokines and chemokines, but also influence the activity of bone marrow derived cells and blood born macrophage populations. In many diseases including brain disorders and spinal cord injury, researchers have tried to harbor the neuroprotective and repair properties of these subpopulations. Hematopoietic bone marrow derived cells (BMDCs) are of great interest, especially during gene therapy because certain hematopoietic cell subpopulations traffic to the sites of injury and inflammation. The aim of this study was to develop a method of labeling endogenous bone marrow derived cells through intraosseous impregnation of recombinant adeno-associated virus (rAAV) or lentivirus. We utilized rAAV serotype 9 (rAAV-9) or lentivirus for gene delivery of green florescence protein (GFP) to the mouse bone marrow cells. Flow cytometry showed that both viruses were able to efficiently transduce mouse bone marrow cells in vivo. However, the rAAV9-GFP viral construct transduced BMDCs more efficiently than the lentivirus (11.2% vs. 6.8%), as indicated by cellular GFP expression. We also demonstrate that GFP labeled cells correspond to bone marrow cells of myeloid origin using CD11b as a marker. Additionally, we characterized the ability of bone marrow derived, GFP labeled cells to extravasate into the brain parenchyma upon acute and subchronic neuroinflammatory stimuli in the mouse CNS. Viral mediated over expression of chemokine (C-C motif) ligand 2 (CCL2) or intracranial injection of lipopolysaccharide (LPS) recruited GFP labeled BMDCs from the periphery into the brain parenchyma compared to vehicle treated mice. Altogether our findings demonstrate a useful method of labeling endogenous BMDCs via viral transduction and the ability to track subpopulations throughout the body

  2. Optimal Labeling Dose, Labeling Time, and Magnetic Resonance Imaging Detection Limits of Ultrasmall Superparamagnetic Iron-Oxide Nanoparticle Labeled Mesenchymal Stromal Cells

    Directory of Open Access Journals (Sweden)

    Anders Bruun Mathiasen

    2013-01-01

    Full Text Available Background. Regenerative therapy is an emerging treatment modality. To determine migration and retention of implanted cells, it is crucial to develop noninvasive tracking methods. The aim was to determine ex vivo magnetic resonance imaging (MRI detection limits of ultrasmall superparamagnetic iron-oxide (USPIO labeled mesenchymal stromal cells (MSCs. Materials and Methods. 248 gel-phantoms were constructed and scanned on a 1.5T MRI-scanner. Phantoms contained human MSCs preincubated with USPIO nanoparticles for 2, 6, or 21 hours using 5 or 10 μg USPIO/105 MSCs. In addition, porcine hearts were scanned after injection of USPIO labeled MSCs. Results. Using 21 h incubation time and 10 μg USPIO/105 MSCs, labeled cells were clearly separated from unlabeled cells on MRI using 250.000 (P<0.001, 500.000 (P=0.007, and 1.000.000 MSCs (P=0.008. At lower incubation times and doses, neither labeled nor unlabeled cells could be separated. In porcine hearts labeled, but not unlabeled, MSCs were identified on MRI. Conclusions. As few as 250.000 MSCs can be detected on MRI using 21 h incubation time and 10 μg USPIO/105 MSCs. At lower incubation times and doses, several million cells are needed for MRI detection. USPIO labeled cells can be visualized by MRI in porcine myocardial tissue.

  3. Synchrotron X-ray fluorescence studies of a bromine-labelled cyclic RGD peptide interacting with individual tumor cells

    International Nuclear Information System (INIS)

    The first example of synchrotron X-ray fluorescence imaging of cultured mammalian cells in cyclic peptide research is reported. The study reports the first quantitative analysis of the incorporation of a bromine-labelled cyclic RGD peptide and its effects on the biodistribution of endogenous elements (for example, K and Cl) within individual tumor cells. The first example of synchrotron X-ray fluorescence imaging of cultured mammalian cells in cyclic peptide research is reported. The study reports the first quantitative analysis of the incorporation of a bromine-labelled cyclic RGD peptide and its effects on the biodistribution of endogenous elements (for example, K and Cl) within individual tumor cells

  4. Ultra-fast stem cell labelling using cationised magnetoferritin

    Science.gov (United States)

    Correia Carreira, S.; Armstrong, J. P. K.; Seddon, A. M.; Perriman, A. W.; Hartley-Davies, R.; Schwarzacher, W.

    2016-03-01

    Magnetic cell labelling with superparamagnetic iron oxide nanoparticles (SPIONs) facilitates many important biotechnological applications, such as cell imaging and remote manipulation. However, to achieve adequate cellular loading of SPIONs, long incubation times (24 hours and more) or laborious surface functionalisation are often employed, which can adversely affect cell function. Here, we demonstrate that chemical cationisation of magnetoferritin produces a highly membrane-active nanoparticle that can magnetise human mesenchymal stem cells (hMSCs) using incubation times as short as one minute. Magnetisation persisted for several weeks in culture and provided significant T2* contrast enhancement during magnetic resonance imaging. Exposure to cationised magnetoferritin did not adversely affect the membrane integrity, proliferation and multi-lineage differentiation capacity of hMSCs, which provides the first detailed evidence for the biocompatibility of magnetoferritin. The combination of synthetic ease and flexibility, the rapidity of labelling and absence of cytotoxicity make this novel nanoparticle system an easily accessible and versatile platform for a range of cell-based therapies in regenerative medicine.Magnetic cell labelling with superparamagnetic iron oxide nanoparticles (SPIONs) facilitates many important biotechnological applications, such as cell imaging and remote manipulation. However, to achieve adequate cellular loading of SPIONs, long incubation times (24 hours and more) or laborious surface functionalisation are often employed, which can adversely affect cell function. Here, we demonstrate that chemical cationisation of magnetoferritin produces a highly membrane-active nanoparticle that can magnetise human mesenchymal stem cells (hMSCs) using incubation times as short as one minute. Magnetisation persisted for several weeks in culture and provided significant T2* contrast enhancement during magnetic resonance imaging. Exposure to cationised

  5. Labeling and imaging cells in the zebrafish hindbrain.

    Science.gov (United States)

    Jayachandran, Pradeepa; Hong, Elim; Brewster, Rachel

    2010-07-25

    Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells (1) . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos (2). The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

  6. Labeling index in squamous cell carcinoma of the larynx

    Energy Technology Data Exchange (ETDEWEB)

    Balzi, M.; Ninu, B.M.; Becciolini, A.; Scubla, E.; Boanini, P.; Gallina, E.; Gallo, O.; Fini-Storchi, O.; Bondi, R. (Radiation Biology Laboratory, University of Florence (Italy))

    1991-07-01

    Two cell kinetic parameters, the 3H-thymidine labeling index (TLI) and the mitotic index (MI), were studied in vitro on fragments of squamous cell carcinoma tissue of the larynx. They were evaluated to identify those elements able to characterize the growth of these solid tumors. The values of these parameters were analyzed as a function of the clinical stage and the involvement of the regional lymph nodes. Results showed a statistically significant increase in the TLI from stage T1 to T3. No statistically significant differences in the TLI values were observed between the patients with positive and negative lymph nodes.

  7. Nutrition labelling: a review of research on consumer and industry response in the global South

    Directory of Open Access Journals (Sweden)

    Jessie Mandle

    2015-01-01

    Full Text Available Background: To identify peer-reviewed research on consumers’ usage and attitudes towards the nutrition label and the food industry's response to labelling regulations outside Europe, North America, and Australia and to determine knowledge gaps for future research. Design: Narrative review. Results: This review identified nutrition labelling research from 20 countries in Asia, Africa, the Middle East, and Latin America. Consumers prefer that pre-packaged food include nutrition information, although there is a disparity between rates of use and comprehension. Consumer preference is for front-of-pack labelling and for information that shows per serving or portion as a reference unit, and label formats with graphics or symbols. Research on the food and beverage industry's response is more limited but shows that industry plays an active role in influencing legislation and regulation. Conclusions: Consumers around the world share preferences with consumers in higher income countries with respect to labelling. However, this may reflect the research study populations, who are often better educated than the general population. Investigation is required into how nutrition labels are received in emerging economies especially among the urban and rural poor, in order to assess the effectiveness of labelling policies. Further research into the outlook of the food and beverage industry, and also on expanded labelling regulations is a priority. Sharing context-specific research regarding labelling between countries in the global South could be mutually beneficial in evaluating obesity prevention policies and strategies.

  8. Efficient preparation and labeling of human induced pluripotent stem cells by nanotechnology

    Directory of Open Access Journals (Sweden)

    Jing Ruan

    2011-02-01

    Full Text Available Jing Ruan1,*, Jie Shen2,*, Zheng Wang2, Jiajia Ji1, Hua Song1, Kan Wang1, Bin Liu1, Jinhui Li2, Daxiang Cui11Department of Bio-Nano Science and Engineering, Key Laboratory for Thin Film and Microfabrication Technology of the Ministry of Education, National Key Laboratory of Micro/Nano Fabrication Technology, Research Institute of Micro/Nano Science and Technology, Shanghai Jiao Tong University, Shanghai, People’s Republic of China; 2Shanghai Institute of Digestive Diseases, Shanghai Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China. *These two authors contributed equally to this workAbstract: Efficient preparation and labeling of human induced pluripotent stem (iPS cells is a great challenge in stem cell research and development. With the aim of investigating the feasibility of using nanotechnology to enhance the preparation efficiency of iPS cells and to label iPS cells for long-term tracing and imaging, in this paper, four transcription factor genes, ie, Oct4, Sox2, LIN28, and Nanog, and packaging plasmids such as PSPAX2 and PMD2.G were cotransfected into 293T cells using Generation 5.0 polyamidoamine dendrimer-modified magnetic nanoparticles (dMNPs as a delivery system. The resultant supernatant liquids were incubated with human fibroblast cells at 37°C for 21 days, then the embryonic stem (ES cell-like clones were screened, cultured, and identified. Finally, the prepared iPS cells were labeled with fluorescent magnetic nanoparticles (FMNPs. The results showed that dMNPs can efficiently deliver all vectors into 293T cells. The resultant lentiviruses’ titers were 10-fold more than those based on Lipofectamine™ 2000. Reverse transcription polymerase chain reaction analysis showed that four genes (Oct4, Sox2, LIN28, and Nanog exhibited different expressions in iPS cells. Immunostaining analysis showed that specific surface markers of ES cells such as SSEA-3, SSEA-4, Tra-1-60, and Tra

  9. Some technetium complexes for labelling red blood cells

    International Nuclear Information System (INIS)

    A new approach to produce technetium labelled red blood cells, used routinely in diagnostic nuclear medicine, is reported. The enzyme Carbonic Anhydrase (CA), present in erythrocytes, is strongly inhibited by primary aromatic sulphonamides, which bind at the enzyme active site. Three types of ligand able to coordinate to technetium and suitable for modification to include a primary aromatic sulphonamide group were studied; bis(thiosemicarbazones), Schiff bases and some propylene amine oximes. The experimental conditions needed to label the ligands were determined. Both the thiosemicarbazone and propyleneamine oxime derivatives were labelled, but under no conditions attempted were the Schiff bases complexed by Technetium. The two major isozymes of Human Carbonic Anhydrase, HCA I and HCA II, were isolated from blood. The strength of binding of the free ligands SET, PN130 and PN135 with each of the isozymes was measured and expressed as the Dissociation Constant Kd. The rate of uptake of the technetium complexes into washed RBCs and whole blood was measured and found to be much slower in whole blood. The biodistribution of both TcPN130 and TcPN135 in rats was determined and scintigraphic images for the TcPN130 complex were recorded. Attempts to synthesise the Tc-99 analogues on the milligram scale to allow chemical characterisation of these complexes were unsuccessful. (author)

  10. Some technetium complexes for labelling red blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Emery, M.F.

    1988-01-01

    A new approach to produce technetium labelled red blood cells, used routinely in diagnostic nuclear medicine, is reported. The enzyme Carbonic Anhydrase (CA), present in erythrocytes, is strongly inhibited by primary aromatic sulphonamides, which bind at the enzyme active site. Three types of ligand able to coordinate to technetium and suitable for modification to include a primary aromatic sulphonamide group were studied; bis(thiosemicarbazones), Schiff bases and some propylene amine oximes. The experimental conditions needed to label the ligands were determined. Both the thiosemicarbazone and propyleneamine oxime derivatives were labelled, but under no conditions attempted were the Schiff bases complexed by Technetium. The two major isozymes of Human Carbonic Anhydrase, HCA I and HCA II, were isolated from blood. The strength of binding of the free ligands SET, PN130 and PN135 with each of the isozymes was measured and expressed as the Dissociation Constant K{sub d}. The rate of uptake of the technetium complexes into washed RBCs and whole blood was measured and found to be much slower in whole blood. The biodistribution of both TcPN130 and TcPN135 in rats was determined and scintigraphic images for the TcPN130 complex were recorded. Attempts to synthesise the Tc-99 analogues on the milligram scale to allow chemical characterisation of these complexes were unsuccessful. (author).

  11. Information on Stem Cell Research

    Science.gov (United States)

    ... Enhancing Diversity Find People About NINDS Information on Stem Cell Research Research @ NINDS Stem Cell Highlights Submit a hESC ... found here: Human Induced Pluripotent Stem Cells NINDS Stem Cell Research on Campus The Intramural Research Program of NINDS ...

  12. Nanoparticle-labeled stem cells: a novel therapeutic vehicle

    Directory of Open Access Journals (Sweden)

    Abir O El-Sadik

    2010-03-01

    Full Text Available Abir O El-Sadik1, Afaf El-Ansary2, Sherif M Sabry31Stem Cell Unit, Anatomy Department, College of Medicine, Health Science Colleges; 2Biochemistry Department, Science College, King Saud University; 3Anatomy Department, Faculty of Medicine, Cairo University, Cairo, EgyptAbstract: Nanotechnology has been described as a general purpose technology. It has already generated a range of inventions and innovations. Development of nanotechnology will provide clinical medicine with a range of new diagnostic and therapeutic opportunities such as medical imaging, medical diagnosis, drug delivery, and cancer detection and management. Nanoparticles such as manganese, polystyrene, silica, titanium oxide, gold, silver, carbon, quantum dots, and iron oxide have received enormous attention in the creation of new types of analytical tools for biotechnology and life sciences. Labeling of stem cells with nanoparticles overcame the problems in homing and fixing stem cells to their desired site and guiding extension of stem cells to specific directions. Although the biologic effects of some nanoparticles have already been assessed, information on toxicity and possible mechanisms of various particle types remains inadequate. The aim of this review is to give an overview of the mechanisms of internalization and distribution of nanoparticles inside stem cells, as well as the influence of different types of nanoparticles on stem cell viability, proliferation, differentiation, and cytotoxicity, and to assess the role of nanoparticles in tracking the fate of stem cells used in tissue regeneration.Keywords: nanoparticles, stem cells, uptake, differentiation, cytotoxicity, tracking

  13. Creation of Primary Cell Lines from Lineage-Labeled Mouse Models of Cancer

    Science.gov (United States)

    Rhim, Andrew D.

    2015-01-01

    Frequently, it is necessary to isolate pure populations of cancer cells for downstream assays, such as transcriptional analysis, signaling studies, and the creation of noncontaminated primary cell lines. Genetic lineage labeling with fluorescent reporter alleles allows for the identification of epithelial-derived cells within tumors. This protocol describes a method to isolate lineage-labeled pancreatic epithelial cells for ex vivo analysis, but it can be adapted for any type of lineage-labeled tumor. PMID:25934932

  14. Creation of Primary Cell Lines from Lineage-Labeled Mouse Models of Cancer

    OpenAIRE

    Rhim, Andrew D.

    2015-01-01

    Frequently, it is necessary to isolate pure populations of cancer cells for downstream assays, such as transcriptional analysis, signaling studies, and the creation of noncontaminated primary cell lines. Genetic lineage labeling with fluorescent reporter alleles allows for the identification of epithelial-derived cells within tumors. This protocol describes a method to isolate lineage-labeled pancreatic epithelial cells for ex vivo analysis, but it can be adapted for any type of lineage-label...

  15. HaloTag protein-mediated specific labeling of living cells with quantum dots

    International Nuclear Information System (INIS)

    Quantum dots emerge as an attractive alternative to small molecule fluorophores as fluorescent tags for in vivo cell labeling and imaging. This communication presents a method for specific labeling of live cells using quantum dots. The labeling is mediated by HaloTag protein expressed at the cell surface which forms a stable covalent adduct with its ligand (HaloTag ligand). The labeling can be performed in one single step with quantum dot conjugates that are functionalized with HaloTag ligand, or in two steps with biotinylated HaloTag ligand first and followed by streptavidin coated quantum dots. Live cell fluorescence imaging indicates that the labeling is specific and takes place at the cell surface. This HaloTag protein-mediated cell labeling method should facilitate the application of quantum dots for live cell imaging

  16. Multiplexed labeling system for high-throughput cell sorting.

    Science.gov (United States)

    Shin, Seung Won; Park, Kyung Soo; Song, In Hyun; Shin, Woo Jung; Kim, Byung Woo; Kim, Dong-Ik; Um, Soong Ho

    2016-09-01

    Flow cytometry and fluorescence activated cell sorting techniques were designed to realize configurable classification and separation of target cells. A number of cell phenotypes with different functionalities have recently been revealed. Before simultaneous selective capture of cells, it is desirable to label different samples with the corresponding dyes in a multiplexing manner to allow for a single analysis. However, few methods to obtain multiple fluorescent colors for various cell types have been developed. Even when restricted laser sources are employed, a small number of color codes can be expressed simultaneously. In this study, we demonstrate the ability to manifest DNA nanostructure-based multifluorescent colors formed by a complex of dyes. Highly precise self-assembly of fluorescent dye-conjugated oligonucleotides gives anisotropic DNA nanostructures, Y- and tree-shaped DNA (Y-DNA and T-DNA, respectively), which may be used as platforms for fluorescent codes. As a proof of concept, we have demonstrated seven different fluorescent codes with only two different fluorescent dyes using T-DNA. This method provides maximum efficiency for current flow cytometry. We are confident that this system will provide highly efficient multiplexed fluorescent detection for bioanalysis compared with one-to-one fluorescent correspondence for specific marker detection. PMID:27181032

  17. Efficient in vitro labeling rabbit neural stem cell with paramagnetic Gd-DTPA and fluorescent substance

    International Nuclear Information System (INIS)

    Objectives: The aim of this study is to label rabbit neural stem cells (NSCs) by using standard contrast agents (Gd-DTPA) in combination with PKH26 and in vitro track them with MR imaging. Materials and methods: NSCs from prenatal brains of rabbits were cultured and propagated. Intracellular uptake of Gd-DTPA was achieved by using a non-liposomal lipid transfection reagent (Effectene) as the transfection agent. After labeling with Gd-DTPA, cells were incubated with cellular membrane fluorescent dye PKH26. The labeling effectiveness and the longevity of Gd-DTPA maintenance were measured on a 1.5 T MR scanner. The influence of labeling on the cellular biological behaviors was assessed by cellular viability, proliferation and differentiation assessment. Results: The labeling efficiency of Gd-DTPA was up to 90%. The signal intensity on T1-weighted imaging and T1 values of labeled cells were significantly higher than those of unlabeled cells (P 3. Cellular uptake of Gd-DTPA was maintained until 15 days after initially labeling. There was no significant difference in the cellular viability and proliferation between the labeled and unlabeled NSCs (P > 0.05). Normal glial and neuronal differentiation remained in labeled NSCs like unlabeled NSCs. Conclusion: Highly efficient labeling NSCs with Gd-DTPA could be achieved by using Effectene. This method of labeling NSCs allows for tracking cells with MR imaging, and without alterations of cellular biological behaviors.

  18. Increasing magnetite contents of polymeric magnetic particles dramatically improves labeling of neural stem cell transplant populations.

    Science.gov (United States)

    Adams, Christopher F; Rai, Ahmad; Sneddon, Gregor; Yiu, Humphrey H P; Polyak, Boris; Chari, Divya M

    2015-01-01

    Safe and efficient delivery of therapeutic cells to sites of injury/disease in the central nervous system is a key goal for the translation of clinical cell transplantation therapies. Recently, 'magnetic cell localization strategies' have emerged as a promising and safe approach for targeted delivery of magnetic particle (MP) labeled stem cells to pathology sites. For neuroregenerative applications, this approach is limited by the lack of available neurocompatible MPs, and low cell labeling achieved in neural stem/precursor populations. We demonstrate that high magnetite content, self-sedimenting polymeric MPs [unfunctionalized poly(lactic acid) coated, without a transfecting component] achieve efficient labeling (≥90%) of primary neural stem cells (NSCs)-a 'hard-to-label' transplant population of major clinical relevance. Our protocols showed high safety with respect to key stem cell regenerative parameters. Critically, labeled cells were effectively localized in an in vitro flow system by magnetic force highlighting the translational potential of the methods used.

  19. Effect of labeling with iron oxide particles or nanodiamonds on the functionality of adipose-derived mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Sinead P Blaber

    Full Text Available Stem cells are increasingly the focus of translational research as well as having emerging roles in human cellular therapy. To support these uses there is a need for improved methods for in vivo cell localization and tracking. In this study, we examined the effects of cell labeling on the in vitro functionality of human adipose-derived mesenchymal stem cells. Our results provide a basis for future in vivo studies investigating implanted cell fate and longevity. In particular, we investigated the effects of two different particles: micron-sized (~0.9 µm fluorescently labeled (Dragon Green superparamagnetic iron oxide particles (M-SPIO particles; and, carboxylated nanodiamonds of ~0.25 µm in size. The effects of labeling on the functionality of adipose-derived MSCs were assessed by in vitro morphology, osteogenic and adipogenic differentiation potential, CD marker expression, cytokine secretion profiling and quantitative proteomics of the intra-cellular proteome. The differentiation and CD marker assays for stem-like functionality were not altered upon label incorporation and no secreted or intra-cellular protein changes indicative of stress or toxicity were detected. These in vitro results indicate that the M-SPIO particles and nanodiamonds investigated in this study are biocompatible with MSCs and therefore would be suitable labels for cell localization and tracking in vivo.

  20. Label-Free Imaging of Umbilical Cord Tissue Morphology and Explant-Derived Cells

    Science.gov (United States)

    Paesen, Rik; Gyselaers, Wilfried; Stinissen, Piet

    2016-01-01

    In situ detection of MSCs remains difficult and warrants additional methods to aid with their characterization in vivo. Two-photon confocal laser scanning microscopy (TPM) and second harmonic generation (SHG) could fill this gap. Both techniques enable the detection of cells and extracellular structures, based on intrinsic properties of the specific tissue and intracellular molecules under optical irradiation. TPM imaging and SHG imaging have been used for label-free monitoring of stem cells differentiation, assessment of their behavior in biocompatible scaffolds, and even cell tracking in vivo. In this study, we show that TPM and SHG can accurately depict the umbilical cord architecture and visualize individual cells both in situ and during culture initiation, without the use of exogenously applied labels. In combination with nuclear DNA staining, we observed a variance in fluorescent intensity in the vessel walls. In addition, antibody staining showed differences in Oct4, αSMA, vimentin, and ALDH1A1 expression in situ, indicating functional differences among the umbilical cord cell populations. In future research, marker-free imaging can be of great added value to the current antigen-based staining methods for describing tissue structures and for the identification of progenitor cells in their tissue of origin. PMID:27746820

  1. Label-Free Imaging of Umbilical Cord Tissue Morphology and Explant-Derived Cells

    Directory of Open Access Journals (Sweden)

    Raf Donders

    2016-01-01

    Full Text Available In situ detection of MSCs remains difficult and warrants additional methods to aid with their characterization in vivo. Two-photon confocal laser scanning microscopy (TPM and second harmonic generation (SHG could fill this gap. Both techniques enable the detection of cells and extracellular structures, based on intrinsic properties of the specific tissue and intracellular molecules under optical irradiation. TPM imaging and SHG imaging have been used for label-free monitoring of stem cells differentiation, assessment of their behavior in biocompatible scaffolds, and even cell tracking in vivo. In this study, we show that TPM and SHG can accurately depict the umbilical cord architecture and visualize individual cells both in situ and during culture initiation, without the use of exogenously applied labels. In combination with nuclear DNA staining, we observed a variance in fluorescent intensity in the vessel walls. In addition, antibody staining showed differences in Oct4, αSMA, vimentin, and ALDH1A1 expression in situ, indicating functional differences among the umbilical cord cell populations. In future research, marker-free imaging can be of great added value to the current antigen-based staining methods for describing tissue structures and for the identification of progenitor cells in their tissue of origin.

  2. Retrograde Labeling of Adult Rat Retinal Ganglion Cells with the Flurogold

    Institute of Scientific and Technical Information of China (English)

    Wei Huang; Yannian Hui; Miaoli Zhang

    2000-01-01

    Purpose: To study the densities and distribution of retinal ganglion cells(RGC) in adult rat retinae with flurogold(FG) labeling retogradely.Methods: FG was injected to the superior colliculi(SC) and dorsal lateral geniculate nuclei (dLGN) in adult rats and the retinae were examined by fluorescence microscopy at various periods of time.Results: FG-labelled RGC were observed in the retina as early as 3 days after application of FG. The labelled cells gradually increased in density, reached 95% of the maximal number on days 7 and the maximal number on days 30. The density of labelled cells was higher in the posterior pole than in the peripheral area. The fluorescence intensity in labelled cells maintained up to 60 days.Conclusion: The FG retrograde labeling method is reliable and effective for quantity of RGC. Eye Science 2000; 16:29 ~ 33.

  3. Retrograde Labeling of Adult Rat Retinal Ganglion Cells with the Flurogold

    Institute of Scientific and Technical Information of China (English)

    WeiHuang; YannianHui; 等

    2002-01-01

    Purpose:To study the densities and distribution of retinal ganglion cells(RGC) in adult rat retinae with flurogold(FG) labeling retogradely.Methods:FG was injected to the superior colliculid(SC) and dorsal lateral geniculate nuclei(dLGN) in adult rats and the retinae were examined by fluorescence microscopy at various periods of time.Results:FG-labelled RGC were observed in the retina as early as 3 days after application of FG.The labeled cells gradually increased in density,reached 95% of the maximal number on days 7 and the maximal nuber on days 30.The density of labeled cells was higher in the posterior pole than in the peripheral area.The fluorescence intensity in labeled cells maintained up to 60 days.Conclusion:The FG retrograde labeling method is reliable and effective for quantity of RGC.Eye Science 2000;46:29-33.

  4. Reception and research of medical and biologic properties labelled tecnetium-99M ciprofloxacin of the hydlochloride

    International Nuclear Information System (INIS)

    Full text: The researches are carried out on creation of a reagent for reception labeled technetium-99m antimicrobial preparations. An estimation of influence of components of a reactionary mix on radiochemical cleanliness received preparations spent a method thinlayer chromatography in various environments. Formation colloid the tin absorbing more of 70 percent of a radioactive label is noted

  5. Detection of the cancer marker CD146 expression in melanoma cells with semiconductor quantum dot label.

    Science.gov (United States)

    Zheng, Hong; Chen, Guangchun; DeLouise, Lisa A; Lou, Ziyang

    2010-08-01

    The use of highly specific and highly sensitive quantum dots immunofluorescent label is a promising approach for biomedical imaging in cancer cells. Human melanoma cell adhesion molecule CD146, overexpressed on the surface of melanoma cells, is an important target for melanoma diagnostics. We synthesized PEG-COOH capped highly fluorescent CdSe/ZnS QDs and conjugated them with streptavidin to prepare QD-SA label. Then, we used QD-SA to link with biotinylated goat anti-mouse IgG and mouse anti-human CD146 to label CD146 overexpressed on live and fixed cells by FACS and Confocal microscopy. Labeling of target cells was shown to have high brightness, photostability, and specificity. Advantages of QD conjugates over FITC conjugates are discussed. The results indicate that construction based on QD-SA label, biotinylated IgG and CD146 antibody can be successfully used for detection of melanoma cells for biomedical applications. PMID:21323102

  6. Preparation of labelled lipids by the use of plant cell cultures

    International Nuclear Information System (INIS)

    The preparation of some radioacitvely labelled lipids by the use of plant cell cultures is discussed and further applications of the new method are suggested. Cell suspension cultures of rape (Brassica napus) and soya (Glycine max) have been used for the preparation of lipids labelled with radioisotopes. Radioactive acetic acid as well as various long-chain fatty acids are readily incorporated into the neutral and ionic lipids of plant cell cultures. In addition, 14C-labelled glycerol, ethanolamine and choline are well utilized by the cells. Randomly labelled lipids have been obtained by incubating cell suspension cultures of rape and soya with [1-14C] acetic acid, and uniformly labelled lipids have been isolated from cultures that had been incubated with a mixture of [1-14C] acetic acid plus [2-14C] acetic acid. The use of techniques of plant cell cultures for the preparation of lipds labelled with stable or radioactive isotopesappears particularly rewarding because the uptake of precursors by the cells and their incorporation into various lipid compounds proceeds rapidly and often quanitatively.This new approach should be useful also for the biosynthesis of lipids whose acyl moieties contain a spn radical, a fluorescent group, or a light-sensitive label. Thus, plant cell cultures constitute valuable new tools for the biosynthetic preparation of a great variety of labelled lipids. (A.G.)

  7. Labeling of human mesenchymal stem cell: Comparison between paramagnetic and superparamagnetic agents

    Science.gov (United States)

    Yang, Chung-Yi; Tai, Ming-Fong; Chen, Shin-Tai; Wang, Yi-Ting; Chen, Ya-Fang; Hsiao, Jong-Kai; Wang, Jaw-Lin; Liu, Hon-Man

    2009-04-01

    Paramagnetic and superparamagnetic substances are used to trace stem cell in living organisms under magnetic resonance imaging (MRI). We compared paramagnetic and superparamagnetic substance for their labeling efficiency by using clinically widely used gadolinium chelates and iron oxide nanoparticles. Without the aid of transfection agent, human mesenchymal stem cells were labeled with each agent separately in different concentration and the optimized concentration was determined by maintaining same cell viability as unlabeled cells. Iron oxide nanoparticle labeling has a detecting threshold of 12 500 cells in vitro, while gadolinium chelates labeling could be detected for at least 50 000 cells. In life animal study, we found there is an eightfold sensitivity in cells labeled with iron oxide superparamagnetic nanoparticles; however, the magnetic susceptibility artifact would obscure the detail of adjacent anatomical structures. We conclude that labeling stem cells with superparamagnetic substance is more efficacious. However, the cells labeled by superparamagnetic nanoparticles might interfere with the interpretation of anatomical structure. These findings would be beneficial to applications of magnetic substances toward stem cell biology and tissue engineering.

  8. Understanding Consumer Behaviour: a Qualitative Research of Eco-Labelling in the Cosmetic Industry

    OpenAIRE

    Pelissier, Camille

    2012-01-01

    This dissertation aims at investigating the way ethical and traditional consumers manage their eco-labelled cosmetics purchases. In this qualitative research, six participants have been interviewed around different topics drawn upon the research questions and the literature review: cosmetics purchases patterns, understanding of the eco-label concept, cosmetics buying criteria and price premium. Then, a discourse analysis method has been used to explore the data collected. The main findings...

  9. Adhesion of endometrial cells labeled with 111 Indium-tropolonate to peritoneum: a novel in vitro model to study endometriosis

    OpenAIRE

    Beliard, Aude; Noël, Agnès; Goffin, Frédéric; Frankenne, Francis; Foidart, Jean-Michel

    2003-01-01

    Objective: To evaluate, in a new original in vitro assay, putative factors that could modulate the adhesion of endometrial cells to peritoneum. Design: Prospective, controlled in vitro study. Setting: Academic research laboratory. Patient(s): Fourteen nonmenopausal women undergoing hysterectomy or laparoscopy for benign gynecologic indication. Intervention(s): Endometrial cells obtained from women with regular cycles without endometriosis were labeled with (111)Indium and confronted in vitro ...

  10. RGDC Peptide Modified Quantum Dots Labelling and Imaging of Tumor Cells

    Institute of Scientific and Technical Information of China (English)

    GUO Yi; LI Chun-rong; SHEN Huai-bin; ZHANG Xue-zhong; LI Lin-song; YU Qian; XU Li

    2011-01-01

    The labelling and imaging of tumor cells were investigated via arginine-glycine-aspartic acidcysteine(RGDC) peptide-labelled quantum dots(QDs).The results show that RGDC modified QDs can label SMMC-7721 tumor cells and adhere to cellular membrane.In constrast,the unmodified QDs are mainly dispersed around the cell.We also found that the RGDC-QDs can penetrate into the cell at 2 h of incubation.After 6 h of incubation,RGDC-QDs can accumulate in a unique intracellular region.

  11. Tracking of CFSE-labeled endothelial progenitor cells in laser-injured mouse retina

    Institute of Scientific and Technical Information of China (English)

    SHI Hui; YANG Wei; CUI Zhi-hua; LU Cheng-wei; LI Xiao-hong; LIANG Ling-ling; SONG E

    2011-01-01

    Background Endothelial progenitor cells (EPCs) transplantation is a promising therapeutic strategy for ischemic retinopathy. The current study aimed to establish a simple, reliable and fluorescent labeling method for tracking EPCs with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) in laser-injured mouse retina.Methods EPCs were isolated from human umbilical cord blood mononuclear cells, cultivated, and labeled with various concentrations of CFSE. Based on fluorescence intensity and cell morphology, a 15 minutes incubation with 5 μmol/L CFSE at 37℃ was selected as the optimal labeling condition. The survival capability and the apoptosis rate of CFSE-labeled EPCs were measured by Trypan blue staining and Annexin V/PI staining assay respectively. Fluorescence microscopy was used to observe the label stability during the extended culture period. Labeled EPCs were transplanted into the vitreous cavity of pigmented mice injured by retinal laser photocoagulation. Evans Blue angiography and flat mounted retinas were examined to track the labeled cells.Results EPCs labeled with 5 μmol/L CFSE presented an intense green fluorescence and maintained normal morphology, with no significant changes in the survival capability or apoptosis rate after being labeled for 2 days, 1 and 4 weeks. The fluorescence intensity gradually decreased in the cells at the end of 4 weeks. Evans Blue angiography of the retina displayed the retinal capillarity network clearly and fluorescence leakage was observed around photocoagulated spots in the laser-injured mouse model. One week after transplantation of labeled EPCs, the fluorescent cells were identified around the photocoagulated lesions. Four weeks after transplantation, fluorescent tube-like structures were observed in the retinal vascular networks.Conclusion EPCs could be labeled by CFSE in vitro and monitored in vivo for at least 4 weeks, and participate in the repair of injured retinal vessels.

  12. MR imaging features of gadofluorine-labeled matrix-associated stem cell implants in cartilage defects.

    Directory of Open Access Journals (Sweden)

    Hossein Nejadnik

    Full Text Available OBJECTIVES: The purpose of our study was to assess the chondrogenic potential and the MR signal effects of GadofluorineM-Cy labeled matrix associated stem cell implants (MASI in pig knee specimen. MATERIALS AND METHODS: Human mesenchymal stem cells (hMSCs were labeled with the micelle-based contrast agent GadofluorineM-Cy. Ferucarbotran-labeled hMSCs, non-labeled hMSCs and scaffold only served as controls. Chondrogenic differentiation was induced and gene expression and histologic evaluation were performed. The proportions of spindle-shaped vs. round cells of chondrogenic pellets were compared between experimental groups using the Fisher's exact test. Labeled and unlabeled hMSCs and chondrocytes in scaffolds were implanted into cartilage defects of porcine femoral condyles and underwent MR imaging with T1- and T2-weighted SE and GE sequences. Contrast-to-noise ratios (CNR between implants and adjacent cartilage were determined and analyzed for significant differences between different experimental groups using the Kruskal-Wallis test. Significance was assigned for p0.017. However, hMSC differentiation into chondrocytes was superior for unlabeled and GadofluorineM-Cy-labeled cells compared with Ferucarbotran-labeled cells, as evidenced by a significantly higher proportion of spindle cells in chondrogenic pellets (p<0.05. GadofluorineM-Cy-labeled hMSCs and chondrocytes showed a positive signal effect on T1-weighted images and a negative signal effect on T2-weighted images while Ferucarbotran-labeled cells provided a negative signal effect on all sequences. CNR data for both GadofluorineM-Cy-labeled and Ferucarbotran-labeled hMSCs were significantly different compared to unlabeled control cells on T1-weighted SE and T2*-weighted MR images (p<0.017. CONCLUSION: hMSCs can be labeled by simple incubation with GadofluorineM-Cy. The labeled cells provide significant MR signal effects and less impaired chondrogenesis compared to Ferucarbotran-labeled h

  13. Labeling embryonic stem cells with enhanced green fluorescent protein on the hypoxanthineguanine phosphoribosyl transferase locus

    Institute of Scientific and Technical Information of China (English)

    滕路; 孟国良; 刑阳; 尚克刚; 王小珂; 顾军

    2003-01-01

    Objective To label embryonic stem (ES) cells with enhanced green fluorescent protein (EGF P) on the hypoxanthineguanine phosphoribosyl transferase (HPRT) gene locus for t he first time to provide a convenient and efficient way for cell tracking and ma nipulation in the studies of transplantation and stem cell therapy.Methods Homologous fragments were obtained by polymerase chain reaction (PCR), from whic h the gene targeting vector pHPRT-EGFP was constructed. The linearized vector was introduced into ES cells by electroporation. The G418r6TGr cell clones were obtained after selection with G418 and 6TG media. The integration patterns of these resistant cell clones were identified with Southern blotting.Results EGFP expressing ES cells on the locus of HPRT were successfu lly generated. They have normal properties, such as karyotype, viability and di fferentiation ability. The green fluorescence of EGFP expressing cells was main tained in propagation of the ES cells for more than 30 passages and in different iated cells. Cultured in suspension, the "green" ES cells aggregated and forme d embryoid bodies, retaining the green fluorescence at varying developmental sta ges. The "green" embryoid bodies could expand and differentiate into various t ypes of cells, exhibiting ubiquitous green fluorescence. Conclusions This generation of "green" targeted ES cells is described in an efficient proto col for obtaining the homologous fragments by PCR. Introducing the marker gene in the genome of ES cells, we should be able to manipulate them in vitro and use them as vehicles in cell-replacement therapy as well as for other biomedical a nd research purposes.

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

    Science.gov (United States)

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

    1993-05-01

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

  15. Optimal labeling dose, labeling time, and magnetic resonance imaging detection limits of ultrasmall superparamagnetic iron-oxide nanoparticle labeled mesenchymal stromal cells

    DEFF Research Database (Denmark)

    Mathiasen, Anders Bruun; Hansen, Louise; Friis, Tina;

    2013-01-01

    Background. Regenerative therapy is an emerging treatment modality. To determine migration and retention of implanted cells, it is crucial to develop noninvasive tracking methods. The aim was to determine ex vivo magnetic resonance imaging (MRI) detection limits of ultrasmall superparamagnetic iron......-oxide (USPIO) labeled mesenchymal stromal cells (MSCs). Materials and Methods. 248 gel-phantoms were constructed and scanned on a 1.5T MRI-scanner. Phantoms contained human MSCs preincubated with USPIO nanoparticles for 2, 6, or 21 hours using 5 or 10  μ g USPIO/10(5) MSCs. In addition, porcine hearts were...

  16. The effect of 51Cr-labelling on cell morphology, in vitro, when evaluating the cytotoxicity of endodontic filling materials

    International Nuclear Information System (INIS)

    Human periodontal ligament fibroblasts and L 929 cell line labelled with chromium 51 were examined byelectron microscope to evaluate the effect of the chromium labeling on the cell ultrastructure. The cells were labeled with chromium 12-20 hours before the start of the experiment. After two and four hours of incubation at 37 degree C and 100% humidity, the cells were examined by scanning and transmission electron microscopy. The result showed that the chromium labeling did not cause any morphological changes. (author)

  17. Simultaneous measurement of NK cell cytotoxicity against two target cell lines labelled with fluorescent lanthanide chelates.

    Science.gov (United States)

    Lövgren, J; Blomberg, K

    1994-07-12

    We describe a cytotoxicity assay which permits the simultaneous measurement of natural killer cell activity against two different cell lines. The target cell lines are labelled either with a fluorescent europium chelate or with a fluorescent terbium chelate and cell death is quantified by measuring the chelate release. K-562, Molt4 and Daudi cell lines have been used as targets. The release of the two chelates from the target cells can be detected with the help of time resolved fluorometry. As the measurements are made after background fluorescence has decayed no additional steps are needed to correct for the background from the medium. The assay procedure used for measurement of cytotoxicity against two target cell lines is very similar to the widely used 51Cr release assay. PMID:8034979

  18. Differentiation of cytotoxicity using target cells labelled with europium and samarium by electroporation.

    Science.gov (United States)

    Bohlen, H; Manzke, O; Engert, A; Hertel, M; Hippler-Altenburg, R; Diehl, V; Tesch, H

    1994-07-12

    We report the simultaneous use of europium-DTPA (Eu-DTPA) and samarium-DTPA (Sm-DTPA) in cytotoxicity experiments to analyze simultaneously LAK and NK cell lysis and to differentiate between specific target lysis and bystander killing. The target cells were either labelled with Eu-DTPA or Sm-DTPA chelates by electroporation, which permits the use of target cell lines or primary leukemic B cells (B-CLL) that cannot be labelled by the conventional dextran-sulphate method. The release of europium and samarium reaches a maximum at comparable time intervals (2-3 h). Due to the shorter counting interval within the samarium window the labelling efficiency is about ten times less efficient compared to europium. Using europium as label for the LAK target Daudi and samarium as label for the NK sensitive cell line K562 the differentiation of LAK versus NK activity can be performed in a single culture assay. Also, the killing of B cells and bystander cells by cytotoxic T cells was analyzed in a system where T cells were redirected to B cells through CD3 x CD19 bispecific antibodies. In fact, no bystander killing was noted when bispecific antibodies were used to bridge cytotoxic T cells to the B cells. This approach provides a simple non-radioactive method for evaluating cytotoxicity against two different cells in a single culture well. PMID:8034986

  19. 111Indium labeling of hepatocytes for analysis of short-term biodistribution of transplanted cells.

    Science.gov (United States)

    Gupta, S; Lee, C D; Vemuru, R P; Bhargava, K K

    1994-03-01

    Hepatocyte transplantation is useful for ex vivo gene therapy and liver repopulation. Methods for hepatic reconstitution have recently been developed but optimization of hepatocyte transplantation systems is necessary. To develop systems for noninvasive assessment of the biodistribution of transplanted cells, we labeled hepatocytes with 111indium-oxine. Our initial studies showed that hepatocytes incorporated 111indium-oxine with an efficiency of approximately 20%. After labeling, cell viability was unchanged and 111indium was present in hepatocytes after overnight culture, as well as after intrasplenic transplantation. Transplanted cells were successfully localized by means of scintigraphic imaging. The scintigraphic patterns of cell distribution were different when hepatocytes were transplanted by means of either spleen or internal jugular vein, which deposit cells into separate vascular beds. Quantitative analysis of the biodistribution of 111indium-labeled hepatocytes indicated that within 2 hr of intrasplenic transplantation, cells were predominantly localized in liver and spleen, and occasionally in lungs. To determine whether the rate of intrasplenic cell injection influenced translocation of hepatocytes, we transplanted cells in normal rats. Despite intrasplenic cell injection at a variety of rates, organ-specific distribution of 111indium-labeled hepatocytes remained unchanged. Labeling with 111indium did not affect long-term survival of transplanted hepatocytes. These results indicate that 111indium-labeling of hepatocytes should greatly assist noninvasive analysis in the short-term of the biodistribution of transplanted hepatocytes. PMID:8119703

  20. HoloMonitor M4: holographic imaging cytometer for real-time kinetic label-free live-cell analysis of adherent cells

    Science.gov (United States)

    Sebesta, Mikael; Egelberg, Peter J.; Langberg, Anders; Lindskov, Jens-Henrik; Alm, Kersti; Janicke, Birgit

    2016-03-01

    Live-cell imaging enables studying dynamic cellular processes that cannot be visualized in fixed-cell assays. An increasing number of scientists in academia and the pharmaceutical industry are choosing live-cell analysis over or in addition to traditional fixed-cell assays. We have developed a time-lapse label-free imaging cytometer HoloMonitorM4. HoloMonitor M4 assists researchers to overcome inherent disadvantages of fluorescent analysis, specifically effects of chemical labels or genetic modifications which can alter cellular behavior. Additionally, label-free analysis is simple and eliminates the costs associated with staining procedures. The underlying technology principle is based on digital off-axis holography. While multiple alternatives exist for this type of analysis, we prioritized our developments to achieve the following: a) All-inclusive system - hardware and sophisticated cytometric analysis software; b) Ease of use enabling utilization of instrumentation by expert- and entrylevel researchers alike; c) Validated quantitative assay end-points tracked over time such as optical path length shift, optical volume and multiple derived imaging parameters; d) Reliable digital autofocus; e) Robust long-term operation in the incubator environment; f) High throughput and walk-away capability; and finally g) Data management suitable for single- and multi-user networks. We provide examples of HoloMonitor applications of label-free cell viability measurements and monitoring of cell cycle phase distribution.

  1. Indium-111 oxine labelling affects the cellular integrity of haematopoietic progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Nowak, Bernd; Reinartz, Patrick; Schaefer, Wolfgang M.; Buell, Ulrich [University Hospital, RWTH Aachen University, Department of Nuclear Medicine, Aachen (Germany); Weber, Christian; Schober, Andreas; Zeiffer, Ute; Liehn, Elisa A.; Hundelshausen, Philipp von [University Hospital, RWTH Aachen University, Department of Molecular Cardiovascular Research, Aachen (Germany)

    2007-05-15

    Cell-based therapy by transplantation of progenitor cells has emerged as a promising development for organ repair, but non-invasive imaging approaches are required to monitor the fate of transplanted cells. Radioactive labelling with {sup 111}In-oxine has been used in preclinical trials. This study aimed to validate {sup 111}In-oxine labelling and subsequent in vivo and ex vivo detection of haematopoietic progenitor cells. Murine haematopoietic progenitor cells (10{sup 6}, FDCPmix) were labelled with 0.1 MBq (low dose) or 1.0 MBq (high dose) {sup 111}In-oxine and compared with unlabelled controls. Cellular retention of {sup 111}In, viability and proliferation were determined up to 48 h after labelling. Labelled cells were injected into the cavity of the left or right cardiac ventricle in mice. Scintigraphic images were acquired 24 h later. Organ samples were harvested to determine the tissue-specific activity. Labelling efficiency was 75 {+-} 14%. Cellular retention of incorporated {sup 111}In after 48 h was 18 {+-} 4%. Percentage viability after 48 h was 90 {+-} 1% (control), 58 {+-} 7% (low dose) and 48 {+-} 8% (high dose) (p<0.0001). Numbers of viable cells after 48 h (normalised to 0 h) were 249 {+-} 51% (control), 42 {+-} 8% (low dose) and 32 {+-} 5% (high dose) (p<0.0001). Cells accumulated in the spleen (86.6 {+-} 27.0% ID/g), bone marrow (59.1 {+-} 16.1% ID/g) and liver (30.3 {+-} 9.5% ID/g) after left ventricular injection, whereas most of the cells were detected in the lungs (42.4 {+-} 21.8% ID/g) after right ventricular injection. Radiolabelling of haematopoietic progenitor cells with {sup 111}In-oxine is feasible, with high labelling efficiency but restricted stability. The integrity of labelled cells is significantly affected, with substantially reduced viability and proliferation and limited migration after systemic transfusion. (orig.)

  2. CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells.

    Science.gov (United States)

    Deng, Wulan; Shi, Xinghua; Tjian, Robert; Lionnet, Timothée; Singer, Robert H

    2015-09-22

    Direct visualization of genomic loci in the 3D nucleus is important for understanding the spatial organization of the genome and its association with gene expression. Various DNA FISH methods have been developed in the past decades, all involving denaturing dsDNA and hybridizing fluorescent nucleic acid probes. Here we report a novel approach that uses in vitro constituted nuclease-deficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated caspase 9 (Cas9) complexes as probes to label sequence-specific genomic loci fluorescently without global DNA denaturation (Cas9-mediated fluorescence in situ hybridization, CASFISH). Using fluorescently labeled nuclease-deficient Cas9 (dCas9) protein assembled with various single-guide RNA (sgRNA), we demonstrated rapid and robust labeling of repetitive DNA elements in pericentromere, centromere, G-rich telomere, and coding gene loci. Assembling dCas9 with an array of sgRNAs tiling arbitrary target loci, we were able to visualize nonrepetitive genomic sequences. The dCas9/sgRNA binary complex is stable and binds its target DNA with high affinity, allowing sequential or simultaneous probing of multiple targets. CASFISH assays using differently colored dCas9/sgRNA complexes allow multicolor labeling of target loci in cells. In addition, the CASFISH assay is remarkably rapid under optimal conditions and is applicable for detection in primary tissue sections. This rapid, robust, less disruptive, and cost-effective technology adds a valuable tool for basic research and genetic diagnosis.

  3. SPIO标记骨髓间充质干细胞和软骨细胞共培养的研究%Research of co-culture of chondrocyts and bone marrow mesenchymal stem cells labeled by SPIO in vitro

    Institute of Scientific and Technical Information of China (English)

    詹兴旺; 姜艳; 王文良

    2011-01-01

    [目的]探讨SPIO标记骨髓间充质干细胞和软骨细胞共培养的可行性,为临床修复关节软骨损伤寻找新途径.[方法] 分离、扩增新西兰大白兔骨髓间充质干细胞(BMSCs)和软骨细胞,根据SPIO标记(50μg/ml)和不同培养环境(共培养、单独培养)分4组,每天在倒置显微镜观察共培养后BMSCs的形态变化,共培养14 d后,免疫组化检测Ⅱ型胶原表达情况,阿利新蓝法检测蛋白多糖(GAG)表达水平.[结果] 共培养7 d后标记的部分BMSCs变圆,14 d时BMSCs形态高度分化与成熟软骨细胞相似,其蛋白多糖和Ⅱ型胶原的基因转录和蛋白表达均增高,明显优于对照组,差异具有显著性意义(P<0.01).[结论]1.软骨细胞微环境能有效诱导BMSCs向软骨细胞分化;2.SPIO可以安全、有效地标记BMSCs.%[ Objective] To investigate the feasibility of co- culture of bone marrow mesenchymal stem cells (BMSCs) labeled by SPIO and chondrocytes so as to provide a new clinical approach for repairing damaged articular cartilage. [ Methods ]BMSCs and chondrocytes were exracted from adult New Zealand white rabbits. These cells were isolated and cultured in different environments (co- culture, single- culture) . The shapes of BMSCs before and after co -culture were observed daily by inverted microscopy. The expression of aggrecan and type Ⅱ collagen in different groups were detected with alcian blue staining and immunohistochemistry at 14 days after co - culture in vitro. [ Results] The shapes of BMSCs became round at 7 days after co -culture in vitro. At 14 days after co - culture with chondrocytes, type Ⅱ collagen and aggrecan were markedly increased. There were significant differences between co - culture group and the other single - culture groups ( P < 0. 01 ) . [ Conclusion ]1. Chondrocytes may provide chondrogenic microenvironment to induce chondrogenic differentiation of BMSCs in vitro efficiently. 2. BMSCs can be marked by SPIO safely and

  4. flowCL: ontology-based cell population labelling in flow cytometry

    Science.gov (United States)

    Courtot, Mélanie; Meskas, Justin; Diehl, Alexander D.; Droumeva, Radina; Gottardo, Raphael; Jalali, Adrin; Taghiyar, Mohammad Jafar; Maecker, Holden T.; McCoy, J. Philip; Ruttenberg, Alan; Scheuermann, Richard H.; Brinkman, Ryan R.

    2015-01-01

    Motivation: Finding one or more cell populations of interest, such as those correlating to a specific disease, is critical when analysing flow cytometry data. However, labelling of cell populations is not well defined, making it difficult to integrate the output of algorithms to external knowledge sources. Results: We developed flowCL, a software package that performs semantic labelling of cell populations based on their surface markers and applied it to labelling of the Federation of Clinical Immunology Societies Human Immunology Project Consortium lyoplate populations as a use case. Conclusion: By providing automated labelling of cell populations based on their immunophenotype, flowCL allows for unambiguous and reproducible identification of standardized cell types. Availability and implementation: Code, R script and documentation are available under the Artistic 2.0 license through Bioconductor (http://www.bioconductor.org/packages/devel/bioc/html/flowCL.html). Contact: rbrinkman@bccrc.ca Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25481008

  5. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry

    Science.gov (United States)

    Niehage, Christian; Karbanová, Jana; Steenblock, Charlotte

    2016-01-01

    Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention. PMID:27490675

  6. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry.

    Science.gov (United States)

    Niehage, Christian; Karbanová, Jana; Steenblock, Charlotte; Corbeil, Denis; Hoflack, Bernard

    2016-01-01

    Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention. PMID:27490675

  7. Tumor-initiating label-retaining cancer cells in human gastrointestinal cancers undergo asymmetric cell division.

    Science.gov (United States)

    Xin, Hong-Wu; Hari, Danielle M; Mullinax, John E; Ambe, Chenwi M; Koizumi, Tomotake; Ray, Satyajit; Anderson, Andrew J; Wiegand, Gordon W; Garfield, Susan H; Thorgeirsson, Snorri S; Avital, Itzhak

    2012-04-01

    Label-retaining cells (LRCs) have been proposed to represent adult tissue stem cells. LRCs are hypothesized to result from either slow cycling or asymmetric cell division (ACD). However, the stem cell nature and whether LRC undergo ACD remain controversial. Here, we demonstrate label-retaining cancer cells (LRCCs) in several gastrointestinal (GI) cancers including fresh surgical specimens. Using a novel method for isolation of live LRCC, we demonstrate that a subpopulation of LRCC is actively dividing and exhibits stem cells and pluripotency gene expression profiles. Using real-time confocal microscopic cinematography, we show live LRCC undergoing asymmetric nonrandom chromosomal cosegregation LRC division. Importantly, LRCCs have greater tumor-initiating capacity than non-LRCCs. Based on our data and that cancers develop in tissues that harbor normal-LRC, we propose that LRCC might represent a novel population of GI stem-like cancer cells. LRCC may provide novel mechanistic insights into the biology of cancer and regenerative medicine and present novel targets for cancer treatment. PMID:22331764

  8. Fuel Cell Research

    Energy Technology Data Exchange (ETDEWEB)

    Weber, Peter M. [Brown University

    2014-03-30

    Executive Summary In conjunction with the Brown Energy Initiative, research Projects selected for the fuel cell research grant were selected on the following criteria: ➢ They should be fundamental research that has the potential to significantly impact the nation’s energy infrastructure. ➢ They should be scientifically exciting and sound. ➢ They should synthesize new materials, lead to greater insights, explore new phenomena, or design new devices or processes that are of relevance to solving the energy problems. ➢ They involve top-caliper senior scientists with a record of accomplishment, or junior faculty with outstanding promise of achievement. ➢ They should promise to yield at least preliminary results within the given funding period, which would warrant further research development. ➢ They should fit into the overall mission of the Brown Energy Initiative, and the investigators should contribute as partners to an intellectually stimulating environment focused on energy science. Based on these criteria, fourteen faculty across three disciplines (Chemistry, Physics and Engineering) and the Charles Stark Draper Laboratory were selected to participate in this effort.1 In total, there were 30 people supported, at some level, on these projects. This report highlights the findings and research outcomes of the participating researchers.

  9. Fuel Cell Research

    Energy Technology Data Exchange (ETDEWEB)

    Weber, Peter M. [Brown University

    2014-03-30

    Executive Summary In conjunction with the Brown Energy Initiative, research Projects selected for the fuel cell research grant were selected on the following criteria: They should be fundamental research that has the potential to significantly impact the nation’s energy infrastructure. They should be scientifically exciting and sound. They should synthesize new materials, lead to greater insights, explore new phenomena, or design new devices or processes that are of relevance to solving the energy problems. They involve top-caliper senior scientists with a record of accomplishment, or junior faculty with outstanding promise of achievement. They should promise to yield at least preliminary results within the given funding period, which would warrant further research development. They should fit into the overall mission of the Brown Energy Initiative, and the investigators should contribute as partners to an intellectually stimulating environment focused on energy science. Based on these criteria, fourteen faculty across three disciplines (Chemistry, Physics and Engineering) and the Charles Stark Draper Laboratory were selected to participate in this effort.1 In total, there were 30 people supported, at some level, on these projects. This report highlights the findings and research outcomes of the participating researchers.

  10. Labeling Adipose-Derived Stem Cells with Hoechst 33342: Usability and Effects on Differentiation Potential and DNA Damage

    Science.gov (United States)

    Schendzielorz, P.; Froelich, K.; Rak, K.; Gehrke, T.; Scherzad, A.; Hagen, R.; Radeloff, A.

    2016-01-01

    Adipose-derived stem cells (ASCs) have been extensively studied in the field of stem cell research and possess numerous clinical applications. Cell labeling is an essential component of various experimental protocols and Hoechst 33342 (H33342) represents a cost-effective and easy methodology for live staining. The purpose of this study was to evaluate the labeling of rat ASCs with two different concentrations of H33342 (0.5 μg/mL and 5 μg/mL), with particular regard to usability, interference with cell properties, and potential DNA damage. Hoechst 33342 used at a low concentration of 0.5 μg/mL did not significantly affect cell proliferation, viability, or differentiation potential of the ASCs, nor did it cause any significant DNA damage as measured by the olive tail moment. High concentrations of 5 μg/mL H33342, however, impaired the proliferation and viability of the ASCs, and considerable DNA damage was observed. Undesirable colabeling of unlabeled cocultivated cells was seen in particular with higher concentrations of H33342, independent of varying washing procedures. Hence, H33342 labeling with lower concentrations represents a usable method, which does not affect the tested cell properties. However, the colabeling of adjacent cells is a drawback of the technique. PMID:27375746

  11. Labeling Adipose-Derived Stem Cells with Hoechst 33342: Usability and Effects on Differentiation Potential and DNA Damage

    Directory of Open Access Journals (Sweden)

    P. Schendzielorz

    2016-01-01

    Full Text Available Adipose-derived stem cells (ASCs have been extensively studied in the field of stem cell research and possess numerous clinical applications. Cell labeling is an essential component of various experimental protocols and Hoechst 33342 (H33342 represents a cost-effective and easy methodology for live staining. The purpose of this study was to evaluate the labeling of rat ASCs with two different concentrations of H33342 (0.5 μg/mL and 5 μg/mL, with particular regard to usability, interference with cell properties, and potential DNA damage. Hoechst 33342 used at a low concentration of 0.5 μg/mL did not significantly affect cell proliferation, viability, or differentiation potential of the ASCs, nor did it cause any significant DNA damage as measured by the olive tail moment. High concentrations of 5 μg/mL H33342, however, impaired the proliferation and viability of the ASCs, and considerable DNA damage was observed. Undesirable colabeling of unlabeled cocultivated cells was seen in particular with higher concentrations of H33342, independent of varying washing procedures. Hence, H33342 labeling with lower concentrations represents a usable method, which does not affect the tested cell properties. However, the colabeling of adjacent cells is a drawback of the technique.

  12. Siloxane Nanoprobes for Labeling and Dual Modality Functional Imaging of Neural Stem Cells.

    Science.gov (United States)

    Addington, Caroline P; Cusick, Alex; Shankar, Rohini Vidya; Agarwal, Shubhangi; Stabenfeldt, Sarah E; Kodibagkar, Vikram D

    2016-03-01

    Cell therapy represents a promising therapeutic for a myriad of medical conditions, including cancer, traumatic brain injury, and cardiovascular disease among others. A thorough understanding of the efficacy and cellular dynamics of these therapies necessitates the ability to non-invasively track cells in vivo. Magnetic resonance imaging (MRI) provides a platform to track cells as a non-invasive modality with superior resolution and soft tissue contrast. We recently reported a new nanoprobe platform for cell labeling and imaging using fluorophore doped siloxane core nanoemulsions as dual modality ((1)H MRI/Fluorescence), dual-functional (oximetry/detection) nanoprobes. Here, we successfully demonstrate the labeling, dual-modality imaging, and oximetry of neural progenitor/stem cells (NPSCs) in vitro using this platform. Labeling at a concentration of 10 μL/10(4) cells with a 40%v/v polydimethylsiloxane core nanoemulsion, doped with rhodamine, had minimal effect on viability, no effect on migration, proliferation and differentiation of NPSCs and allowed for unambiguous visualization of labeled NPSCs by (1)H MR and fluorescence and local pO2 reporting by labeled NPSCs. This new approach for cell labeling with a positive contrast (1)H MR probe has the potential to improve mechanistic knowledge of current therapies, and guide the design of future cell therapies due to its clinical translatability.

  13. Scattering pulse of label free fine structure cells to determine the size scale of scattering structures

    Science.gov (United States)

    Zhang, Lu; Chen, Xingyu; Zhang, Zhenxi; Chen, Wei; Zhao, Hong; Zhao, Xin; Li, Kaixing; Yuan, Li

    2016-04-01

    Scattering pulse is sensitive to the morphology and components of each single label-free cell. The most direct detection result, label free cell's scattering pulse is studied in this paper as a novel trait to recognize large malignant cells from small normal cells. A set of intrinsic scattering pulse calculation method is figured out, which combines both hydraulic focusing theory and small particle's scattering principle. Based on the scattering detection angle ranges of widely used flow cytometry, the scattering pulses formed by cell scattering energy in forward scattering angle 2°-5° and side scattering angle 80°-110° are discussed. Combining the analysis of cell's illuminating light energy, the peak, area, and full width at half maximum (FWHM) of label free cells' scattering pulses for fine structure cells with diameter 1-20 μm are studied to extract the interrelations of scattering pulse's features and cell's morphology. The theoretical and experimental results show that cell's diameter and FWHM of its scattering pulse agree with approximate linear distribution; the peak and area of scattering pulse do not always increase with cell's diameter becoming larger, but when cell's diameter is less than about 16 μm the monotone increasing relation of scattering pulse peak or area with cell's diameter can be obtained. This relationship between the features of scattering pulse and cell's size is potentially a useful but very simple criterion to distinguishing malignant and normal cells by their sizes and morphologies in label free cells clinical examinations.

  14. Functional endothelial cells derived from embryonic stem cells labeled with HIV transactivator peptide-conjugated superparamagnetic nanoparticles

    Institute of Scientific and Technical Information of China (English)

    GAO Bin; FU Wei-guo; DONG Zhi-hui; FANG Zheng-dong; LIU Zhen-jie; SI Yi; ZHANG Xiang-man; WANG Yu-qi

    2011-01-01

    Background The development of regenerative therapies using derivatives of embryonic stem (ES) cells would be facilitated by a non-invasive method to monitor transplanted cells in vivo,for example,magnetic resonance imaging of cells labeled with superparamagnetic iron oxide (SPIO) nanoparticles.Although ES cells have been labeled with SPIO particles,the potential adverse effects of the label have not been fully examined.The objective of this study was to determine whether SPIO labeling affects murine ES cell viability,proliferation,or ability to differentiate into functional endothelial cells (ECs).Methods Cross-linked iron oxide (CLIO,an SPIO) was conjugated with human immunodeficiency virus transactivator of transcription (HIV-Tat) peptides,and murine ES cells were labeled with either CLiO-Tat,CLIO,or HIV-Tat.After labeling,ES cells were cultured for 4 days and FIk-1+ ES cells identified and sorted by immunocytochemistry and fluorescence activated cell sorting (FACS).FIk-1+ cells were raplated on fibronectin-coated dishes,and ECs were obtained by culturing these for 4 weeks in endothelial cell growth medium supplemented with vascular endothelial growth factor (VEGF).ES cell viability was determined using trypan blue exclusion,and the proportion of SPIO+ cells was evaluated using Prussian blue staining and transmission electron microscopy.After differentiation,the behavior and phenotype of ECs were analyzed by reverse transcription-polymerase chain reaction,flow cytometry,immunocytochemistry,Dil-labeled acetylated low-density lipoprotein (AcLDL) uptake,and Matrigel tube formation assay.Results CLIO-Tat was a highly effective label for ES cells,with >96% of cells incorporating the particles,and it did not alter the viability of the labeled cells.ECs derived from CLIO-Tat+ ES cells were very similar to murine aortic ECs in their morphology,expression of endothelial cell markers,ability to form vascular-like channels,and scavenging of AcLDL from the culture medium

  15. Explicit kinetic heterogeneity: mathematical models for interpretation of deuterium labeling of heterogeneous cell populations.

    Directory of Open Access Journals (Sweden)

    Vitaly V Ganusov

    2010-02-01

    Full Text Available Estimation of division and death rates of lymphocytes in different conditions is vital for quantitative understanding of the immune system. Deuterium, in the form of deuterated glucose or heavy water, can be used to measure rates of proliferation and death of lymphocytes in vivo. Inferring these rates from labeling and delabeling curves has been subject to considerable debate with different groups suggesting different mathematical models for that purpose. We show that the three most common models, which are based on quite different biological assumptions, actually predict mathematically identical labeling curves with one parameter for the exponential up and down slope, and one parameter defining the maximum labeling level. By extending these previous models, we here propose a novel approach for the analysis of data from deuterium labeling experiments. We construct a model of "kinetic heterogeneity" in which the total cell population consists of many sub-populations with different rates of cell turnover. In this model, for a given distribution of the rates of turnover, the predicted fraction of labeled DNA accumulated and lost can be calculated. Our model reproduces several previously made experimental observations, such as a negative correlation between the length of the labeling period and the rate at which labeled DNA is lost after label cessation. We demonstrate the reliability of the new explicit kinetic heterogeneity model by applying it to artificially generated datasets, and illustrate its usefulness by fitting experimental data. In contrast to previous models, the explicit kinetic heterogeneity model 1 provides a novel way of interpreting labeling data; 2 allows for a non-exponential loss of labeled cells during delabeling, and 3 can be used to describe data with variable labeling length.

  16. Lentivirus-mediated bifunctional cell labeling for in vivo melanoma study.

    Science.gov (United States)

    Day, Chi-Ping; Carter, John; Bonomi, Carrie; Esposito, Dominic; Crise, Bruce; Ortiz-Conde, Betty; Hollingshead, Melinda; Merlino, Glenn

    2009-06-01

    Lentiviral vectors (LVs) are capable of labeling a broad spectrum of cell types, achieving stable expression of transgenes. However, for in vivo studies, the duration of marker gene expression has been highly variable. We have developed a series of LVs harboring different promoters for expressing reporter gene in mouse cells. Long-term culture and colony formation of several LV-labeled mouse melanoma cells showed that promoters derived from mammalian house-keeping genes, especially those encoding RNA polymerase II (Pol2) and ferritin (FerH), provided the highest consistency for reporter expression. For in vivo studies, primary B16BL6 mouse melanoma were infected with LVs whose luciferase-green fluorescence protein fusion gene (Luc/GFP) was driven by either Pol2 or FerH promoters. When transplanted into syngeneic C57BL/6 mice, Luc/GFP-labeled B16BL6 mouse melanoma cells can be monitored by bioluminescence imaging in vivo, and GFP-positive cells can be isolated from the tumors by fluorescence-activated cell sorter. Pol2-Luc/GFP labeling, while lower in activity, was more sustainable than FerH-Luc/GFP labeling in B16BL6 over consecutive passages into mice. We conclude that Pol-2-Luc/GFP labeling allows long-term in vivo monitoring and tumor cell isolation in immunocompetent mouse melanoma models. PMID:19175523

  17. Measurement of cell mediated cytotoxicity by post-labeling surviving target cells

    International Nuclear Information System (INIS)

    The 51Cr release assay (CRA) is the commonly accepted technique for measurement of cell mediated cytotoxicity. This assay shows some disadvantages when mononucleated cells of human peripheral blood (MNC) are used as effector and target cells. The uptake of 51Cr by PHA stimulated lymphocytes is low compared to the spontaneous release. In an attempt to develop a cytotoxicity assay suitable for human lymphocytes we used 14C-TdR to label target cells surviving after contact with effector cells. Cytotoxic lymphocytes were generated by incubation of MNC with irradiated allogeneic MNC for 6 days. On day 6 the effector cells are irradiated and cocultured with PHA stimulated target cells. Twenty-four hours later 14C-TdR is added. After an additional 24 h the cultures are harvested and 14C-TdR taken up by target cells is measured. It is shown that the effector cells are still cytotoxic after irradiation. These cells do not take up 14C-TdR. Cell-free supernatants do not influence the uptake of 14C-TdR by target cells. The results obtained with this assay correlate very well those obtained by the CRA, if the spontaneous release does not exceed 30%. (author)

  18. Magnetic targeting of iron-oxide-labeled fluorescent hepatoma cells to the liver

    Energy Technology Data Exchange (ETDEWEB)

    Luciani, Alain [Universite Rene Descartes, Hopital Europeen Georges Pompidou, Laboratoire de Recherche en Imagerie, EA 4062, Paris (France); Imagerie Medicale, Faculte de Medecine Paris XII, CHU Henri Mondor, Creteil cedex (France); Wilhelm, Claire; Gazeau, Florence [Universite Paris Diderot, Batiment Condorcet, Laboratoire Matiere et Systemes Complexes, CNRS-UMR 7057, Paris Cedex (France); Bruneval, Patrick [Anatomopathologie, Hopital Europeen Georges Pompidou, Paris (France); Cunin, Patrick [Unite de Recherche Clinique, Faculte de Medecine Paris XII, CHU Henri Mondor, Creteil cedex (France); Autret, Gwennhael; Clement, Olivier [Universite Rene Descartes, Hopital Europeen Georges Pompidou, Laboratoire de Recherche en Imagerie, EA 4062, Paris (France); Rahmouni, Alain [Imagerie Medicale, Faculte de Medecine Paris XII, CHU Henri Mondor, Creteil cedex (France)

    2009-05-15

    The purpose of this study was to determine whether an external magnet field can induce preferential trafficking of magnetically labeled Huh7 hepatoma cells to the liver following liver cell transplantation. Huh7 hepatoma cells were labeled with anionic magnetic nanoparticles (AMNP) and tagged with a fluorescent membrane marker (PKH67). Iron-uptake was measured by magnetophoresis. Twenty C57Bl6 mice received an intrasplenic injection of 2 x 10{sup 6} labeled cells. An external magnet (0.29 T; 25 T/m) was placed over the liver of 13 randomly selected animals (magnet group), while the remaining 7 animals served as controls. MRI (1.5 T) and confocal fluorescence microscopy (CFM) were performed 10 days post-transplantation. The presence and location of labeled cells within the livers were compared in the magnet group and controls, and confronted with histological analysis representing the standard of reference. Mean iron content per cell was 6 pg. Based on histology, labeled cells were more frequently present within recipient livers in the magnet group (p < 0.01) where their distribution was preferentially peri-vascular (p<0.05). MRI and CFM gave similar results for the overall detection of transplanted cells (kappa=0.828) and for the identification of peri-vascular cells (kappa=0.78). Application of an external magnet can modify the trafficking of transplanted cells, especially by promoting the formation of perivascular aggregates. (orig.)

  19. In vivo quantification of SPIO nanoparticles for cell labeling based on MR phase gradient images.

    Science.gov (United States)

    Wang, Luning; Potter, William M; Zhao, Qun

    2015-01-01

    Along with the development of modern imaging technologies, contrast agents play increasingly important roles in both clinical applications and scientific research. Super-paramagnetic iron oxide (SPIO) nanoparticles, a negative contrast agent, have been extensively used in magnetic resonance imaging (MRI), such as in vivo labeling and tracking of cells. However, there still remain many challenges, such as in vivo quantification of SPIO nanoparticles. In this work, an MR phase gradient-based method was proposed to quantify the SPIO nanoparticles. As a calibration, a phantom experiment using known concentrations (10, 25, 50, 100, 150 and 250 µg/ml) of SPIO was first conducted to verify the proposed quantification method. In a following in vivo experiment, C6 glioma cells labeled with SPIO nanoparticles were implanted into flanks of four mice, which were scanned 1-3 days post-injection for in vivo quantification of SPIO concentration. The results showed that the concentration of SPIO nanoparticles could be determined in both phantom and in vivo experiments using the developed MR phase gradients approach.

  20. Measuring cell cycle progression kinetics with metabolic labeling and flow cytometry.

    Science.gov (United States)

    Fleisig, Helen; Wong, Judy

    2012-01-01

    metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research. PMID:22665142

  1. Stable isotope labelling with amino acids in cell culture for human embryonic stem cell proteomic analysis

    DEFF Research Database (Denmark)

    Harkness, Linda; Prokhorova, Tatyana A; Kassem, Moustapha;

    2012-01-01

    The identification and quantitative measurements of proteins in human embryonic stem cells (hESC) is a fast growing interdisciplinary area with an enormous impact on understanding the biology of hESC and the mechanism controlling self-renewal and differentiation. Using a quantitative mass...... spectroscopic method of stable isotope labelling with amino acids during cell culture (SILAC), we are able to analyse differential expression of proteins from different cellular compartments and to identify intracellular signalling pathways involved in self-renewal and differentiation. In this chapter, we...

  2. Advances in stem cell research

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    @@In 1998, biologists Thomson and Gearhart successfully derived stem cells from human embryos. One year later, several researchers discovered that adult stem cells still retain the ability to be differentiated into unrelated types of cells. Advances in stem cell research open a promising direction for applied medical science. Moreover, it may also force scientists to reconsider the fundamental theory about how cells grow up. Stem cell research was considered by Science as the top of the ten breakthroughs of science of the year[1]. This paper gives a survey of recent advances in stem cell research. 1 Overview In the 1980s, embryonic stem cell and/or embryonic germ cell line (ES cell line, EG cell line) of multifarious mammalian animals, especially those of non-human pri-mates, had been established. In 1998, Thomson and Shamblott obtained ES, EG cell lines from human blasto-cysts and gonad ridges of early human embryos, respec-tively. Their research brought up an ethical debate about whether human embryos can be used as experimental materials. It was not appeased until 1999 when research-ers discovered that stem cells from adults still retain the ability to become different kinds of tissue cells. For in-stance, brain cells can become blood cells[2], and cells from bone marrow can become cells in liver. Scientists believe, for a long time, that cells can only be developed from early pluripotent embryo cells; the differentiation potential of stem cells from mature tissues is restricted to only one of the cell types of the tissue where stem cells are obtained. Recent stem cell researches, however, sub-verted the traditional view of stem cells. These discoveries made scientists speed ahead with the work on adult stem cells, hoping to discover whether their promise will rival that of ES cells.

  3. AGE AND SEX CHARACTERISTICS OF MELATONIN-POSITIVE-LABELED CELLS OF THE GASTRIC MUCOSA IN DESYNCHRONOSIS IN RATS.

    Science.gov (United States)

    Hnatiuk, V; Kononenko, N; Kozub, T; Chikitkina, V; Galiy, L

    2016-06-01

    The aim of the research was to study the state of melatonin-positive-labeled cells (MPLC) of GM in desynchronosis in rats of different age and gender. 780 sections of the pyloric part of the gastric mucosa were studied in rats of both genders at the age of 9, 15 and 20 months. Animals were divided into intact control groups and the groups of the animals kept under the conditions of continuous light for 14 days - desynchronosis. The study was performed by the method of immunohistochemical staining with the primary antibodies to melatonin (Biorbyt, UK) and the secondary Alexa Fluor 488-conjugated antibody (Abcam, UK). In the course of the research it was found that MPLC in all experimental groups were mainly located in the basal and middle segments of the tubular glands of gastric mucosa and were represented by three types of cells. In desynchronosis the number of melatonin-positive-labeled cells significantly reduced in almost every age group, with the exception of females at the age of 20 months. Thus in elderly males and females the number of melatonin-positive-labeled cells of type III increases, whereas in young and mature males it decreases, and cells of type I predominate. PMID:27441544

  4. Supernova: A Versatile Vector System for Single-Cell Labeling and Gene Function Studies in vivo

    Science.gov (United States)

    Luo, Wenshu; Mizuno, Hidenobu; Iwata, Ryohei; Nakazawa, Shingo; Yasuda, Kosuke; Itohara, Shigeyoshi; Iwasato, Takuji

    2016-01-01

    Here we describe “Supernova” series of vector systems that enable single-cell labeling and labeled cell-specific gene manipulation, when introduced by in utero electroporation (IUE) or adeno-associated virus (AAV)-mediated gene delivery. In Supernova, sparse labeling relies on low TRE leakage. In a small population of cells with over-threshold leakage, initial tTA-independent weak expression is enhanced by tTA/TRE-positive feedback along with a site-specific recombination system (e.g., Cre/loxP, Flpe/FRT). Sparse and bright labeling by Supernova with little background enables the visualization of the morphological details of individual neurons in densely packed brain areas such as the cortex and hippocampus, both during development and in adulthood. Sparseness levels are adjustable. Labeled cell-specific gene knockout was accomplished by introducing Cre/loxP-based Supernova vectors into floxed mice. Furthermore, by combining with RNAi, TALEN, and CRISPR/Cas9 technologies, IUE-based Supernova achieved labeled cell-specific gene knockdown and editing/knockout without requiring genetically altered mice. Thus, Supernova system is highly extensible and widely applicable for single-cell analyses in complex organs, such as the mammalian brain. PMID:27775045

  5. In vivo ultrasound and photoacoustic monitoring of mesenchymal stem cells labeled with gold nanotracers.

    Directory of Open Access Journals (Sweden)

    Seung Yun Nam

    Full Text Available Longitudinal monitoring of cells is required in order to understand the role of delivered stem cells in therapeutic neovascularization. However, there is not an imaging technique that is capable of quantitative, longitudinal assessment of stem cell behaviors with high spatial resolution and sufficient penetration depth. In this study, in vivo and in vitro experiments were performed to demonstrate the efficacy of ultrasound-guided photoacoustic (US/PA imaging to monitor mesenchymal stem cells (MSCs labeled with gold nanotracers (Au NTs. The Au NT labeled MSCs, injected intramuscularly in the lower limb of the Lewis rat, were detected and spatially resolved. Furthermore, our quantitative in vitro cell studies indicate that US/PA imaging is capable of high detection sensitivity (1×10⁴ cells/mL of the Au NT labeled MSCs. Finally, Au NT labeled MSCs captured in the PEGylated fibrin gel system were imaged in vivo, as well as in vitro, over a one week time period, suggesting that longitudinal cell tracking using US/PA imaging is possible. Overall, Au NT labeling of MSCs and US/PA imaging can be an alternative approach in stem cell imaging capable of noninvasive, sensitive, quantitative, longitudinal assessment of stem cell behaviors with high spatial and temporal resolutions at sufficient depths.

  6. The use of radiolabeled monoclonal antibodies for cell labeling in vivo

    International Nuclear Information System (INIS)

    The authors have evaluated the potential of in vivo cell surface labeling using radiolabeled monoclonal antibodies (MoAbs) directed against their surface antigens. Two MoAbs, a specific antibody (anti-Thy-1 OX7) and a nonspecific control antibody (anti-CEA) were coupled with DTPA, labeled with /sup 111/In and evaluated against rat thymocytes, marrow cells, and lymphoma cells (all known to be Thy-1 positive) both in vitro and in vivo. Enumeration of the cells which bound the radiolabeled MoAb was done by detecting the antibody on the cell surface with a Fl-F(ab')/sub 2/ goat anti-mouse IgG and analyzing fluorescence (F1) in a flow cytometer (FACS). The thymocytes, which could be labeled in whole blood, showed a labeling efficiency of 80-100%. The labeling, which could be inhibited by cold antibody, was stable up to 72 hours and did not interfere with either cell viability or functional integrity. Following IV injection of the MoAbs in normal rats, there was very good visualization of the bone marrow not seen with the control. Analysis of the marrow cells on the FACS showed that at two hours over 60% of the marrow cells were specifically labeled as against 2% for the control. Within 15 minutes of injecting /sup 111/In-OX7 into rats with lymphoma, 70% of the activity in blood was bound to circulating lymphoma cells. The ability to stably label, rapidly target, and image specific cell populations in vivo has wide ranging diagnostic and therapeutic implications

  7. Controllable labelling of stem cells with a novel superparamagnetic iron oxide-loaded cationic nanovesicle for MR imaging

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Ruo Mi; Zhang, Fang; Wen, Xue Hua; Shen, Jun [Sun Yat-Sen University, Department of Radiology, Sun Yat-Sen Memorial Hospital, Guangzhou, Guangdong (China); Cao, Nuo; Wang, Yi Ru; Shuai, Xin Tao [Sun Yat-Sen University, BME Center, School of Chemistry and Chemical Engineering, Guangzhou (China)

    2012-11-15

    To investigate the feasibility of highly efficient and controllable stem cell labelling for cellular MRI. A new class of cationic, superparamagnetic iron oxide nanoparticle (SPION)-loaded nanovesicles was synthesised to label rat bone marrow mesenchymal stem cells without secondary transfection agents. The optimal labelling conditions and controllability were assessed, and the effect of labelling on cell viability, proliferation activity and multilineage differentiation was determined. In 18 rats, focal ischaemic cerebral injury was induced and the rats randomly injected with 1 x 10{sup 6} cells labelled with 0-, 8- or 20-mV nanovesicles (n = 6 each). In vivo MRI was performed to follow grafted cells in contralateral striata, and results were correlated with histology. Optimal cell labelling conditions involved a concentration of 3.15 {mu}g Fe/mL nanovesicles with 20-mV positive charge and 1-h incubation time. Labelling efficiency showed linear change with an increase in the electric potentials of nanovesicles. Labelling did not affect cell viability, proliferation activity or multilineage differentiation capacity. The distribution and migration of labelled cells could be detected by MRI. Histology confirmed that grafted cells retained the label and remained viable. Stem cells can be effectively and safely labelled with cationic, SPION-loaded nanovesicles in a controllable way for cellular MRI. (orig.)

  8. Labeling and Imaging Mesenchymal Stem Cells with Quantum Dots

    Science.gov (United States)

    Mesenchymal stem cells (MSCs) are multipotent cells with the potential to differentiate into bone, cartilage, adipose and muscle cells. Adult derived MSCs are being actively investigated because of their potential to be utilized for therapeutic cell-based transplantation. Methods...

  9. Fate of 3H-thymidine labelled myogenic cells in regeneration of muscle isografts.

    Science.gov (United States)

    Gutmann, E; Mares, V; Stichová, J

    1976-03-01

    Intact and denervated extensor digitorum longus (EDL) muscles of 20-day-old inbred Lewis-Wistar rats were labelled with 3H-thymidine. Ninety minutes after the injection of the isotope 4.0% of the nuclei were labelled in the intact (i.e. innervated) and 9.6% in the muscles, denervated 3 days before administration of the isotope. The labelled EDL muscles were grafted into the bed of the previously removed EDL muscles of inbred animals and these isografts were studied 30 days later. In the EDL muscles, regenerated from innervated isografts only occasionally labelled endothelial cells were found whereas in the muscles regenerated from denervated isografts also parenchymal muscle nuclei were regularly labelled. The incidence of labelled nuclei in the regenerated EDL muscles was, however, about 20 times lower than in the donor EDL muscles. The presen experiments provide a direct proof of utilization of donor satelite cell nuclei for regeneration in grafted muscle tissue. With respect to the low incidence of labelled nuclei in regenerated EDL muscles, other sources of cells apparently also contribute to the regeneration process.

  10. Tracking of iron-labeled human neural stem cells by magnetic resonance imaging in cell replacement therapy for Parkinson’s disease

    Institute of Scientific and Technical Information of China (English)

    Milagros Ramos-Gmez; Alberto Martnez-Serrano

    2016-01-01

    Human neural stem cells (hNSCs) derived from the ventral mesencephalon are powerful research tools and candidates for cell therapies in Parkinson’s disease. However, their clinical translation has not been fully realized due, in part, to the limited ability to track stem cell regional localization and survival over long periods of time afterin vivo transplantation. Magnetic resonance imaging provides an excellent non-invasive method to study the fate of transplanted cellsin vivo. For magnetic resonance imaging cell tracking, cells need to be labeled with a contrast agent, such as magnetic nanoparticles, at a concentration high enough to be easily detected by magnetic resonance imaging. Grafting of human neural stem cells labeled with magnetic nanoparticles allows cell tracking by magnetic resonance imaging without impairment of cell survival, prolif-eration, self-renewal, and multipotency. However, the results reviewed here suggest that in long term grafting, activated microglia and macrophages could contribute to magnetic resonance imaging signal by engulifng dead labeled cells or iron nanoparticles dispersed freely in the brain parenchyma over time.

  11. A trend analysis of private label research between 2000 and 2012

    OpenAIRE

    Molinillo Jiménez, Sebastián; Ekinci, Yuksel; Whyatt, Georgina; Occhiocupo, Nicolettta

    2014-01-01

    The aim of this study is to review the state of empirical and theoretical research about Private Label (PL) and to identify gaps and future research avenues. This paper uses a bibliometric approach of recent advances in the research of PL; it focuses on the period of greatest scientific output (2000-2012) and includes all international publications on PL in marketing journals ranked by the Academic Journal Quality Guide. The analysis systematically considers main authors and universities, cou...

  12. The migration of synthetic magnetic nanoparticle labeled dendritic cells into lymph nodes with optical imaging

    Directory of Open Access Journals (Sweden)

    Su H

    2013-10-01

    Full Text Available Hang Su,1,* Yongbin Mou,1,* Yanli An,2 Wei Han,1 Xiaofeng Huang,1 Guohua Xia,3 Yanhong Ni,1 Yu Zhang,4 Jianmin Ma,1 Qingang Hu1,5 1Center Laboratory of Stomatology, Stomatological Hospital Affiliated Medical School, Nanjing University, Nanjing, People's Republic of China; 2Jiangsu Key Lab of Molecular and Function Imaging, Department of Radiology; 3Department of Hematology, Zhongda Hospital, Medical School, 4State Key Laboratory of Molecule and Bimolecular Electronics, Jiangsu Provincial Laboratory for Biomaterials and Devices; Southeast University, Nanjing, People's Republic of China; 5Leeds Dental Institute, Faculty of Medicine and health, University of Leeds, Leeds, United Kingdom*These authors contributed equally to this workBackground: The successful biotherapy of carcinoma with dendritic cell (DC vaccines pivotally relies on DCs’ migratory capability into lymph tissues and activation of T cells. Accurate imaging and evaluation of DC migration in vivo have great significance during antitumor treatment with DC vaccine. We herein examined the behavior of DCs influenced by synthetic superparamagnetic iron oxide (SPIO nanoparticle labeling.Methods: γ-Fe2O3 nanoparticles were prepared and DCs, which were induced from bone marrow monocytes of enhanced green fluorescent protein (EGFP transgenic mice, were labeled. The endocytosis of the SPIO, surface molecules, cell apoptosis and fluorescence intensity of EGFP-DCs were displayed by Prussian blue staining and flow cytometry (FCM, respectively. After EGFP-DCs, labeled with SPIO, were injected into footpads (n = 5 for 24 hours, the mice were examined in vivo by optical imaging (OPI. Meanwhile, confocal imaging and FCM were applied, respectively, to detect the migration of labeled DCs into draining lymph nodes.Results: Nearly 100% of cells were labeled by the SPIO, in which the intracellular blue color gradually deepened and the iron contents rose with the increase of labeling iron concentrations

  13. In vivo MRI discrimination between live and lysed iron-labelled cells using balanced steady state free precession

    International Nuclear Information System (INIS)

    The goal of this study was to evaluate the ability of balanced steady state free precession (b-SSFP) magnetic resonance imaging sequence to distinguish between live and lysed iron-labelled cells. Human breast cancer cells were labelled with iron oxide nanoparticles. Cells were lysed using sonication. Imaging was performed at 3 T. The timing parameters for b-SSFP and the number of iron-labelled cells in samples were varied to optimise the b-SSFP signal difference between live and lysed iron-labelled cell samples. For in vivo experiments, cells were mixed with Matrigel and implanted into nude mice. Three mice implanted with live labelled cancer cells were irradiated to validate this method. Lysed iron-labelled cells have a significantly higher signal compared with live, intact iron-labelled cells in bSSFP images. The contrast between live and dead cells can be maximised by careful optimisation of timing parameters. A change in the b-SSFP signal was measured 6 days after irradiation, reflecting cell death in vivo. Histology confirmed the presence of dead cells in the implant. Our results show that the b-SSFP sequence can be optimised to allow for the discrimination of live iron-labelled cells and lysed iron-labelled cells in vitro and in vivo. (orig.)

  14. Self-Assembled Superparamagnetic Iron Oxide Nanoclusters for Universal Cell Labeling and MRI

    Science.gov (United States)

    Chen, Shuzhen; Zhang, Jun; Jiang, Shengwei; Lin, Gan; Luo, Bing; Yao, Huan; Lin, Yuchun; He, Chengyong; Liu, Gang; Lin, Zhongning

    2016-05-01

    Superparamagnetic iron oxide (SPIO) nanoparticles have been widely used in a variety of biomedical applications, especially as contrast agents for magnetic resonance imaging (MRI) and cell labeling. In this study, SPIO nanoparticles were stabilized with amphiphilic low molecular weight polyethylenimine (PEI) in an aqueous phase to form monodispersed nanocomposites with a controlled clustering structure. The iron-based nanoclusters with a size of 115.3 ± 40.23 nm showed excellent performance on cellular uptake and cell labeling in different types of cells, moreover, which could be tracked by MRI with high sensitivity. The SPIO nanoclusters presented negligible cytotoxicity in various types of cells as detected using MTS, LDH, and flow cytometry assays. Significantly, we found that ferritin protein played an essential role in protecting stress from SPIO nanoclusters. Taken together, the self-assembly of SPIO nanoclusters with good magnetic properties provides a safe and efficient method for universal cell labeling with noninvasive MRI monitoring capability.

  15. Labeling human embryonic stem-cell-derived cardiomyocytes for tracking with MR imaging

    Energy Technology Data Exchange (ETDEWEB)

    Castaneda, Rosalinda T.; Daldrup-Link, Heike [Lucile Packard Children' s Hospital, Stanford School of Medicine, Pediatric Radiology, Stanford, CA (United States); Boddington, Sophie; Wendland, Mike; Mandrussow, Lydia [University of California, Department of Radiology and Biomedical Imaging, UCSF Medical Center, San Francisco, CA (United States); Henning, Tobias D. [University Hospital of Cologne, Department of Radiology and Neuroradiology, Cologne (Germany); Liu, Siyuan [National Institutes of Health, Language Section, Voice, Speech and Language Branch, National Institute on Deafness and Other Communication Disorders, Bethesda, MD (United States)

    2011-11-15

    Human embryonic stem cells (hESC) can generate cardiomyocytes (CM), which offer promising treatments for cardiomyopathies in children. However, challenges for clinical translation result from loss of transplanted cell from target sites and high cell death. An imaging technique that noninvasively and repetitively monitors transplanted hESC-CM could guide improvements in transplantation techniques and advance therapies. To develop a clinically applicable labeling technique for hESC-CM with FDA-approved superparamagnetic iron oxide nanoparticles (SPIO) by examining labeling before and after CM differentiation. Triplicates of hESC were labeled by simple incubation with 50 {mu}g/ml of ferumoxides before or after differentiation into CM, then imaged on a 7T MR scanner using a T2-weighted multi-echo spin-echo sequence. Viability, iron uptake and T2-relaxation times were compared between groups using t-tests. hESC-CM labeled before differentiation demonstrated significant MR effects, iron uptake and preserved function. hESC-CM labeled after differentiation showed no significant iron uptake or change in MR signal (P < 0.05). Morphology, differentiation and viability were consistent between experimental groups. hESC-CM should be labeled prior to CM differentiation to achieve a significant MR signal. This technique permits monitoring delivery and engraftment of hESC-CM for potential advancements of stem cell-based therapies in the reconstitution of damaged myocardium. (orig.)

  16. Metabolic labeling of Caenorhabditis elegans primary embryonic cells with azido-sugars as a tool for glycoprotein discovery.

    Directory of Open Access Journals (Sweden)

    Amanda R Burnham-Marusich

    Full Text Available Glycobiology research with Caenorhabditis elegans (C. elegans has benefitted from the numerous genetic and cell biology tools available in this system. However, the lack of a cell line and the relative inaccessibility of C. elegans somatic cells in vivo have limited the biochemical approaches available in this model. Here we report that C. elegans primary embryonic cells in culture incorporate azido-sugar analogs of N-acetylgalactosamine (GalNAc and N-acetylglucosamine (GlcNAc, and that the labeled glycoproteins can be analyzed by mass spectrometry. By using this metabolic labeling approach, we have identified a set of novel C. elegans glycoprotein candidates, which include several mitochondrially-annotated proteins. This observation was unexpected given that mitochondrial glycoproteins have only rarely been reported, and it suggests that glycosylation of mitochondrially-annotated proteins might occur more frequently than previously thought. Using independent experimental strategies, we validated a subset of our glycoprotein candidates. These include a mitochondrial, atypical glycoprotein (ATP synthase α-subunit, a predicted glycoprotein (aspartyl protease, ASP-4, and a protein family with established glycosylation in other species (actin. Additionally, we observed a glycosylated isoform of ATP synthase α-subunit in bovine heart tissue and a primate cell line (COS-7. Overall, our finding that C. elegans primary embryonic cells are amenable to metabolic labeling demonstrates that biochemical studies in C. elegans are feasible, which opens the door to labeling C. elegans cells with other radioactive or azido-substrates and should enable the identification of additional post-translationally modified targets and analysis of the genes required for their modification using C. elegans mutant libraries.

  17. Variability of the thymidine labeling index in squamous cell carcinoma of the head and neck

    Energy Technology Data Exchange (ETDEWEB)

    Greenberg, B.; Woo, L.; Blatchford, S.; Aguirre, M.; Garewal, H.

    1988-06-01

    Tritiated thymidine (/sup 3/HTdR) labeling is the standard technique for determining the kinetic activity of tumors. This method has been used to label multiple sections of tumor specimens obtained from seven patients with advanced squamous cell carcinoma of the head and neck. Considerable variability was observed in the labeling index in different sites from the same specimen. To reduce the large sampling error due to heterogeneity, we recommend that an average value be determined from multiple sections when employing this technique.

  18. Magnetic resonance imaging of ultrasmall superparamagnetic iron oxide-labeled exosomes from stem cells: a new method to obtain labeled exosomes

    Science.gov (United States)

    Busato, Alice; Bonafede, Roberta; Bontempi, Pietro; Scambi, Ilaria; Schiaffino, Lorenzo; Benati, Donatella; Malatesta, Manuela; Sbarbati, Andrea; Marzola, Pasquina; Mariotti, Raffaella

    2016-01-01

    Purpose Recent findings indicate that the beneficial effects of adipose stem cells (ASCs), reported in several neurodegenerative experimental models, could be due to their paracrine activity mediated by the release of exosomes. The aim of this study was the development and validation of an innovative exosome-labeling protocol that allows to visualize them with magnetic resonance imaging (MRI). Materials and methods At first, ASCs were labeled using ultrasmall superparamagnetic iron oxide nanoparticles (USPIO, 4–6 nm), and optimal parameters to label ASCs in terms of cell viability, labeling efficiency, iron content, and magnetic resonance (MR) image contrast were investigated. Exosomes were then isolated from labeled ASCs using a standard isolation protocol. The efficiency of exosome labeling was assessed by acquiring MR images in vitro and in vivo as well as by determining their iron content. Transmission electron microscopy images and histological analysis were performed to validate the results obtained. Results By using optimized experimental parameters for ASC labeling (200 µg Fe/mL of USPIO and 72 hours of incubation), it was possible to label 100% of the cells, while their viability remained comparable to unlabeled cells; the detection limit of MR images was of 102 and 2.5×103 ASCs in vitro and in vivo, respectively. Exosomes isolated from previously labeled ASCs retain nanoparticles, as demonstrated by transmission electron microscopy images. The detection limit by MRI was 3 µg and 5 µg of exosomes in vitro and in vivo, respectively. Conclusion We report a new approach for labeling of exosomes by USPIO that allows detection by MRI while preserving their morphology and physiological characteristics. PMID:27330291

  19. Subcellular SIMS imaging of isotopically labeled amino acids in cryogenically prepared cells

    Energy Technology Data Exchange (ETDEWEB)

    Chandra, Subhash

    2004-06-15

    Ion microscopy is a potentially powerful technique for localization of isotopically labeled molecules. In this study, L-arginine and phenylalanine amino acids labeled with stable isotopes {sup 13}C and {sup 15}N were localized in cultured cells with the ion microscope at 500 nm spatial resolution. Cells were exposed to the labeled amino acids and cryogenically prepared. SIMS analyses were made in fractured freeze-dried cells. A dynamic distribution was observed from labeled arginine-treated LLC-PK{sub 1} kidney cells at mass 28 ({sup 13}C{sup 15}N) in negative secondaries, revealing cell-to-cell heterogeneity and preferential accumulation of the amino acid (or its metabolite) in the nucleus and nucleolus of some cells. The smaller nucleolus inside the nucleus was clearly resolved in SIMS images and confirmed by correlative light microscopy. The distribution of labeled phenylalanine contrasted with arginine as it was rather homogeneously distributed in T98G human glioblastoma cells. Images of {sup 39}K, {sup 23}Na and {sup 40}Ca were also recorded to confirm the reliability of sample preparation and authenticity of the observed amino acid distributions. These observations indicate that SIMS techniques can provide a valuable technology for subcellular localization of nitrogen-containing molecules in proteomics since nitrogen does not have a radionuclide tracer isotope. Amino acids labeled with stable isotopes can be used as tracers for studying their transport and metabolism in distinct subcellular compartments with SIMS. Further studies of phenylalanine uptake in human glioblastoma cells may have special significance in boron neutron capture therapy (BNCT) as a boron analogue of phenylalanine, boronophenylalanine is a clinically approved compound for the treatment of brain tumors.

  20. Subcellular SIMS imaging of isotopically labeled amino acids in cryogenically prepared cells

    Science.gov (United States)

    Chandra, Subhash

    2004-06-01

    Ion microscopy is a potentially powerful technique for localization of isotopically labeled molecules. In this study, L-arginine and phenylalanine amino acids labeled with stable isotopes 13C and 15N were localized in cultured cells with the ion microscope at 500 nm spatial resolution. Cells were exposed to the labeled amino acids and cryogenically prepared. SIMS analyses were made in fractured freeze-dried cells. A dynamic distribution was observed from labeled arginine-treated LLC-PK 1 kidney cells at mass 28 ( 13C15N) in negative secondaries, revealing cell-to-cell heterogeneity and preferential accumulation of the amino acid (or its metabolite) in the nucleus and nucleolus of some cells. The smaller nucleolus inside the nucleus was clearly resolved in SIMS images and confirmed by correlative light microscopy. The distribution of labeled phenylalanine contrasted with arginine as it was rather homogeneously distributed in T98G human glioblastoma cells. Images of 39K, 23Na and 40Ca were also recorded to confirm the reliability of sample preparation and authenticity of the observed amino acid distributions. These observations indicate that SIMS techniques can provide a valuable technology for subcellular localization of nitrogen-containing molecules in proteomics since nitrogen does not have a radionuclide tracer isotope. Amino acids labeled with stable isotopes can be used as tracers for studying their transport and metabolism in distinct subcellular compartments with SIMS. Further studies of phenylalanine uptake in human glioblastoma cells may have special significance in boron neutron capture therapy (BNCT) as a boron analogue of phenylalanine, boronophenylalanine is a clinically approved compound for the treatment of brain tumors.

  1. Tc-99m Labeled HMPAO white Blood Cell Scintigraphy in Pediatric Patients

    OpenAIRE

    Funda Aydın; Arzu Kın Cengiz; Fırat Güngör

    2012-01-01

    Objective: 99mTc labeled hexamethylpropylene amine oxime (HMPAO) white blood cell (WBC) scintigraphy is a frequently used option for acute infection, particularly in pediatric patients. This scintigraphy is applied to detect sites of infection/inflammation in patients with fever of unknown origin, to find and follow up osteomyelitis, and to detect suspicion of acute appendicitis. The aim of this retrospective study was to evaluate the value of 99mTc-HMPAO labeled WBC scintigraphy in pediatric...

  2. MRI of magnetically labeled mesenchymal stem cells in hepatic failure model

    Institute of Scientific and Technical Information of China (English)

    Kyu; Ri; Son; Se; Young; Chung; Hyo-Cheol; Kim; Hoe; Suk; Kim; Seung; Hong; Choi; Jeong; Min; Lee; Woo; Kyung; Moon

    2010-01-01

    AIM:To track intravascularly transplanted mesenchymal stem cells (MSCs) labeled with superparamagnetic iron oxide (SPIO) by using magnetic resonance imaging (MRI) in an experimental rabbit model of hepatic failure.METHODS:Human MSCs labeled with FDA-approved SPIO particles (Feridex) were transplanted via the mes-enteric vein into rabbits (n=16) with carbon tetrachloride-induced hepatic failure.Magnetic resonance (MR) examinations were performed with a 3.0 T clinical scanner immediately before and 2 h and 1,...

  3. Functional investigations on embryonic stem cells labeled with clinically translatable iron oxide nanoparticles

    Science.gov (United States)

    Liu, Jing; Wang, Liqin; Cao, Jianbo; Huang, Yue; Lin, Yu; Wu, Xiaoyun; Wang, Zhiyong; Zhang, Fan; Xu, Xiuqin; Liu, Gang

    2014-07-01

    Stem cell based therapies offer significant potential in the field of regenerative medicine. The development of superparamagnetic iron oxide (SPIO) nanoparticle labeling and magnetic resonance imaging (MRI) have been increasingly used to track the transplanted cells, enabling in vivo determination of cell fate. However, the impact of SPIO-labeling on the cell phenotype and differentiation capacity of embryonic stem cells (ESCs) remains unclear. In this study, we wrapped SPIO nanoparticles with stearic acid grafted PEI600, termed as Stearic-LWPEI-SPIO, to generate efficient and non-toxic ESC labeling tools. Our results showed that efficient labeling of ESCs at an optimized low dosage of Stearic-LWPEI-SPIO nanoparticles did not alter the differentiation and self-renewal properties of ESCs. The localization of the transplanted ESCs observed by MRI correlated well with histological studies. These findings demonstrate that Stearic-LWPEI-SPIO nanoparticles have potential to be clinically translatable MRI probes and may enable non-invasive in vivo tracking of ESCs in experimental and clinical settings during cell-based therapies.Stem cell based therapies offer significant potential in the field of regenerative medicine. The development of superparamagnetic iron oxide (SPIO) nanoparticle labeling and magnetic resonance imaging (MRI) have been increasingly used to track the transplanted cells, enabling in vivo determination of cell fate. However, the impact of SPIO-labeling on the cell phenotype and differentiation capacity of embryonic stem cells (ESCs) remains unclear. In this study, we wrapped SPIO nanoparticles with stearic acid grafted PEI600, termed as Stearic-LWPEI-SPIO, to generate efficient and non-toxic ESC labeling tools. Our results showed that efficient labeling of ESCs at an optimized low dosage of Stearic-LWPEI-SPIO nanoparticles did not alter the differentiation and self-renewal properties of ESCs. The localization of the transplanted ESCs observed by MRI

  4. Labeling cells in microtiter plates for determination of [3H]thymidine uptake.

    Science.gov (United States)

    Shevach, E M

    2001-05-01

    A number of protocols in Current Protocols in Immunology use as their end-point the determination of cell proliferation by determining the incorporation of [(3)H]thymidine into cellular DNA. This appendix presents a protocol in which the radioactive label is added during the last 4 to 24 hr of the culture. A semiautomated cell harvesting apparatus is then used to lyse the cells with water and precipitate the labeled DNA on glass fiber filters. The filter pads are then dried and counted by standard liquid scintillation counting techniques in a scintillation counter. PMID:18432656

  5. Mesenchymal stem cell in vitro labeling by hybrid fluorescent magnetic polymeric particles for application in cell tracking.

    Science.gov (United States)

    Supokawej, Aungkura; Nimsanor, Natakarn; Sanvoranart, Tanwarat; Kaewsaneha, Chariya; Hongeng, Suradej; Tangboriboonrat, Pramuan; Jangpatarapongsa, Kulachart

    2015-12-01

    Mesenchymal stem cells (MSCs) are a type of adult stem cell that contains multi-differentiation and proliferative properties and that shows high treatment implications for many clinical problems. The outcome of stem cell transplantation is still limited due to many factors, especially their survival and their interaction with the microenvironment after transplantation. Molecular imaging is a challenging technique that has been used to overcome this limitation and is based on the concept of labeling cells with tractable, visible, and non-toxic materials to track the cells after transplantation. In this study, magnetic polymeric nanoparticles (MPNPs) were used to directly label Wharton's jelly-derived MSCs (WJ-MSCs). After labeling, the growth rate and the viability of the MSCs as well as the time of exposure were determined. The 3D images of WJ-MSCs labeled with MPNPs for 24 h were created using confocal microscopy. The results showed that, after incubation with fluorescent MPNPs for over 8 h, the growth rate and cell viability of the WJ-MSCs was similar to those of the control. Three-dimensional imaging revealed that the fluorescent MPNPs could infiltrate into the cells and spread into the cytoplasm, which suggests that the synthesized fluorescent MPNPs could possibly label MSCs for cell tracking study and be further developed for in vivo applications. PMID:25893425

  6. Near-infrared imaging of adoptive immune cell therapy in breast cancer model using cell membrane labeling.

    Directory of Open Access Journals (Sweden)

    Fatma M Youniss

    Full Text Available The overall objective of this study is to non-invasively image and assess tumor targeting and retention of directly labeled T-lymphocytes following their adoptive transfer in mice. T-lymphocytes obtained from draining lymph nodes of 4T1 (murine breast cancer cell sensitized BALB/C mice were activated in-vitro with Bryostatin/Ionomycin for 18 hours, and were grown in the presence of Interleukin-2 for 6 days. T-lymphocytes were then directly labeled with 1,1-dioctadecyltetramethyl indotricarbocyanine Iodide (DiR, a lipophilic near infrared fluorescent dye that labels the cell membrane. Assays for viability, proliferation, and function of labeled T-lymphocytes showed that they were unaffected by DiR labeling. The DiR labeled cells were injected via tail vein in mice bearing 4T1 tumors in the flank. In some cases labeled 4T1 specific T-lymphocytes were injected a week before 4T1 tumor cell implantation. Multi-spectral in vivo fluorescence imaging was done to subtract the autofluorescence and isolate the near infrared signal carried by the T-lymphocytes. In recipient mice with established 4T1 tumors, labeled 4T1 specific T-lymphocytes showed marked tumor retention, which peaked 6 days post infusion and persisted at the tumor site for up to 3 weeks. When 4T1 tumor cells were implanted 1-week post-infusion of labeled T-lymphocytes, T-lymphocytes responded to the immunologic challenge and accumulated at the site of 4T1 cell implantation within two hours and the signal persisted for 2 more weeks. Tumor accumulation of labeled 4T1 specific T-lymphocytes was absent in mice bearing Meth A sarcoma tumors. When lysate of 4T1 specific labeled T-lymphocytes was injected into 4T1 tumor bearing mice the near infrared signal was not detected at the tumor site. In conclusion, our validated results confirm that the near infrared signal detected at the tumor site represents the DiR labeled 4T1 specific viable T-lymphocytes and their response to immunologic challenge

  7. Selective amine labeling of cell surface proteins guided by coiled-coil assembly.

    Science.gov (United States)

    Yano, Yoshiaki; Furukawa, Nami; Ono, Satoshi; Takeda, Yuki; Matsuzaki, Katsumi

    2016-11-01

    Covalent labeling of target proteins in living cells is useful for both fluorescence live-cell imaging and the subsequent biochemical analyses of the proteins. Here, we report an efficient method for the amine labeling of membrane proteins on the cell surface, guided by a noncovalent coiled-coil interaction. A carboxyl sulfosuccinimidyl ester introduced at the C-terminus of the coiled-coil probe reacted with target proteins under mild labeling conditions ([probe] = 150 nM, pH 7.4, 25°C) for 20 min. Various fluorescent moieties with different hydrophobicities are available for covalent labeling with high signal/background labeling ratios. Using this method, oligomeric states of glycophorin A (GpA) were compared in mammalian CHO-K1 cells and sodium dodecyl sulfate (SDS) micelles. In the cell membranes, no significant self-association of GpA was detected, whereas SDS-PAGE suggested partial dimerization of the proteins. Membrane cholesterol was found to be an important factor that suppressed the dimerization of GpA. Thus, the covalent functionality enables direct comparison of the oligomeric state of membrane proteins under various conditions. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 484-490, 2016. PMID:26285787

  8. Macrophage phagocytosis alters the MRI signal of ferumoxytol-labeled mesenchymal stromal cells in cartilage defects

    Science.gov (United States)

    Nejadnik, Hossein; Lenkov, Olga; Gassert, Florian; Fretwell, Deborah; Lam, Isaac; Daldrup-Link, Heike E.

    2016-05-01

    Human mesenchymal stem cells (hMSCs) are a promising tool for cartilage regeneration in arthritic joints. hMSC labeling with iron oxide nanoparticles enables non-invasive in vivo monitoring of transplanted cells in cartilage defects with MR imaging. Since graft failure leads to macrophage phagocytosis of apoptotic cells, we evaluated in vitro and in vivo whether nanoparticle-labeled hMSCs show distinct MR signal characteristics before and after phagocytosis by macrophages. We found that apoptotic nanoparticle-labeled hMSCs were phagocytosed by macrophages while viable nanoparticle-labeled hMSCs were not. Serial MRI scans of hMSC transplants in arthritic joints of recipient rats showed that the iron signal of apoptotic, nanoparticle-labeled hMSCs engulfed by macrophages disappeared faster compared to viable hMSCs. This corresponded to poor cartilage repair outcomes of the apoptotic hMSC transplants. Therefore, rapid decline of iron MRI signal at the transplant site can indicate cell death and predict incomplete defect repair weeks later. Currently, hMSC graft failure can be only diagnosed by lack of cartilage defect repair several months after cell transplantation. The described imaging signs can diagnose hMSC transplant failure more readily, which could enable timely re-interventions and avoid unnecessary follow up studies of lost transplants.

  9. Label-Free Quantitative Proteomics and N-terminal Analysis of Human Metastatic Lung Cancer Cells

    OpenAIRE

    Min, Hophil; Han, Dohyun; Kim, Yikwon; Cho, Jee Yeon; Jin, Jonghwa; Kim, Youngsoo

    2014-01-01

    Proteomic analysis is helpful in identifying cancer-associated proteins that are differentially expressed and fragmented that can be annotated as dysregulated networks and pathways during metastasis. To examine meta-static process in lung cancer, we performed a proteomics study by label-free quantitative analysis and N-terminal analysis in 2 human non-small-cell lung cancer cell lines with disparate metastatic potentials—NCI-H1703 (primary cell, stage I) and NCI-H1755 (metastatic cell, stage ...

  10. flowCL: ontology-based cell population labelling in flow cytometry

    OpenAIRE

    Courtot, Mélanie; Meskas, Justin; Diehl, Alexander D.; Droumeva, Radina; Gottardo, Raphael; Jalali, Adrin; Taghiyar, Mohammad Jafar; Maecker, Holden T; McCoy, J. Philip; Ruttenberg, Alan; Scheuermann, Richard H.; Brinkman, Ryan R

    2014-01-01

    Motivation: Finding one or more cell populations of interest, such as those correlating to a specific disease, is critical when analysing flow cytometry data. However, labelling of cell populations is not well defined, making it difficult to integrate the output of algorithms to external knowledge sources.

  11. Discrimination of bromodeoxyuridine labelled and unlabelled mitotic cells in flow cytometric bromodeoxyuridine/DNA analysis

    DEFF Research Database (Denmark)

    Jensen, P O; Larsen, J K; Christensen, I J;

    1994-01-01

    Bromodeoxyuridine (BrdUrd) labelled and unlabelled mitotic cells, respectively, can be discriminated from interphase cells using a new method, based on immunocytochemical staining of BrdUrd and flow cytometric four-parameter analysis of DNA content, BrdUrd incorporation, and forward and orthogona...

  12. Identification of miRNA targets with stable isotope labeling by amino acids in cell culture

    DEFF Research Database (Denmark)

    Vinther, Jeppe; Hedegaard, Mads Marquardt; Gardner, Paul Phillip;

    2006-01-01

    miRNAs are small noncoding RNAs that regulate gene expression. We have used stable isotope labeling by amino acids in cell culture (SILAC) to investigate the effect of miRNA-1 on the HeLa cell proteome. Expression of 12 out of 504 investigated proteins was repressed by miRNA-1 transfection. This ...

  13. Laser capture microdissection and genetic analysis of carbon-labeled Kupffer cells

    Institute of Scientific and Technical Information of China (English)

    Stephan Gehring; Edmond Sabo; Maryann E San Martin; Elizabeth M Dickson; Chao-Wen Cheng; Stephen H Gregory

    2009-01-01

    AIM: To develop a method of labeling and microdissecting mouse Kupffer cells within an extraordinarily short period of time using laser capture microdissection (LCM). METHODS: Tissues are complex structures comprised of a heterogeneous population of interconnected cells. LCM offers a method of isolating a single cell type from specific regions of a tissue section. LCM is an essential approach used in conjunction with molecular analysis to study the functional interaction of cells in their native tissue environment. The process of labeling and acquiring cells by LCM prior to mRNA isolation can be elaborate, thereby subjecting the RNA to considerable degradation. Kupffer cell labeling is achieved by injecting India ink intravenously, thus circumventing the need for in vitro staining. The significance of this novel approach was validated using a cholestatic liver injury model. RESULTS: mRNA extracted from the microdissected cell population displayed marked increases in colonystimulating factor-1 receptor and Kupffer cell receptor message expression, which demonstrated Kupffer cell enrichment. Gene expression by Kupffer cells derived from bile-duct-ligated, versus sham-operated, mice was compared. Microarray analysis revealed a significant (2.5-fold, q value < 10) change in 493 genes. Based on this fold-change and a standardized PubMed search, 10 genes were identified that were relevant to the ability of Kupffer cells to suppress liver injury.CONCLUSION: The methodology outlined herein provides an approach to isolating high quality RNA from Kupffer cells, without altering the tissue integrity.

  14. In vivo phenotypic characterisation of nucleoside label-retaining cells in mouse periosteum

    Directory of Open Access Journals (Sweden)

    HM Cherry

    2014-03-01

    Full Text Available Periosteum is known to contain cells that, after isolation and culture-expansion, display properties of mesenchymal stromal/stem cells (MSCs. However, the equivalent cells have not been identified in situ mainly due to the lack of specific markers. Postnatally, stem cells are slow-cycling, long-term nucleoside-label-retaining cells. This study aimed to identify and characterise label-retaining cells in mouse periosteum in vivo. Mice received iodo-deoxy-uridine (IdU via the drinking water for 30 days, followed by a 40-day washout period. IdU+ cells were identified by immunostaining in conjunction with MSC and lineage markers. IdU-labelled cells were detected throughout the periosteum with no apparent focal concentration, and were negative for the endothelial marker von Willebrand factor and the pan-haematopoietic marker CD45. Subsets of IdU+ cells were positive for the mesenchymal/stromal markers vimentin and cadherin-11. IdU+ cells expressed stem cell antigen-1, CD44, CD73, CD105, platelet-derived growth factor receptor-α and p75, thereby displaying an MSC-like phonotype. Co-localisation was not detectable between IdU and the pericyte markers CD146, alpha smooth muscle actin or NG2, nor did IdU co-localise with β-galactosidase in a transgenic mouse expressing this reporter gene in pericytes and smooth muscle cells. Subsets of IdU+ cells expressed the osteoblast-lineage markers Runx2 and osteocalcin. The IdU+ cells expressing osteocalcin were lining the bone and were negative for the MSC marker p75. In conclusion, mouse periosteum contains nucleoside-label-retaining cells with a phenotype compatible with MSCs that are distinct from pericytes and osteoblasts. Future studies characterising the MSC niche in vivo could reveal novel therapeutic targets for promoting bone regeneration/repair.

  15. Liver Label Retaining Cancer Cells Are Relatively Resistant to the Reported Anti-Cancer Stem Cell Drug Metformin

    OpenAIRE

    Xin, Hong-Wu; Ambe, Chenwi M.; Miller, Tyler C.; Chen, Jin-Qiu; Wiegand, Gordon W.; Anderson, Andrew J.; Ray, Satyajit; Mullinax, John E.; Hari, Danielle M; Koizumi, Tomotake; Godbout, Jessica D.; Goldsmith, Paul K.; Stojadinovic, Alexander; Rudloff, Udo; Thorgeirsson, Snorri S.

    2016-01-01

    Background & Aims: Recently, we reported that liver Label Retaining Cancer Cells (LRCC) can initiate tumors with only 10 cells and are relatively resistant to the targeted drug Sorafenib, a standard of practice in advanced hepatocellular carcinoma (HCC). LRCC are the only cancer stem cells (CSC) isolated alive according to a stem cell fundamental function, asymmetric cell division. Metformin has been reported to preferentially target many other types of CSC of different organs, including live...

  16. Stem cell research in China

    OpenAIRE

    Liao, Lianming; Li, Lingsong; Zhao, Robert Chunhua

    2007-01-01

    In the past 5 years, China has increased its efforts in the field of stem cell research and practice. Basic research mainly focuses on bone marrow and embryonic stem cells. Clinical applications of stem cells in the treatment of acute heart failure, acute liver failure and lower limb ischaemia have been reported by many hospitals. China enacted its ‘Ethical Guidelines for Human Embryonic Stem Cell Research’ in 2003. At present, China has the most liberal and favourable environments for human ...

  17. Label-free imaging of gold nanoparticles in single live cells by photoacoustic microscopy

    Science.gov (United States)

    Tian, Chao; Qian, Wei; Shao, Xia; Xie, Zhixing; Cheng, Xu; Liu, Shengchun; Cheng, Qian; Liu, Bing; Wang, Xueding

    2016-03-01

    Gold nanoparticles (AuNPs) have been extensively explored as a model nanostructure in nanomedicine and have been widely used to provide advanced biomedical research tools in diagnostic imaging and therapy. Due to the necessity of targeting AuNPs to individual cells, evaluation and visualization of AuNPs in the cellular level is critical to fully understand their interaction with cellular environment. Currently imaging technologies, such as fluorescence microscopy and transmission electron microscopy all have advantages and disadvantages. In this paper, we synthesized AuNPs by femtosecond pulsed laser ablation, modified their surface chemistry through sequential bioconjugation, and targeted the functionalized AuNPs with individual cancer cells. Based on their high optical absorption contrast, we developed a novel, label-free imaging method to evaluate and visualize intracellular AuNPs using photoacoustic microscopy (PAM). Preliminary study shows that the PAM imaging technique is capable of imaging cellular uptake of AuNPs in vivo at single-cell resolution, which provide an important tool for the study of AuNPs in nanomedicine.

  18. A scalable label-free approach to separate human pluripotent cells from differentiated derivatives.

    Science.gov (United States)

    Willoughby, N A; Bock, H; Hoeve, M A; Pells, S; Williams, C; McPhee, G; Freile, P; Choudhury, D; De Sousa, P A

    2016-01-01

    The broad capacity of pluripotent human embryonic stem cells (hESC) to grow and differentiate demands the development of rapid, scalable, and label-free methods to separate living cell populations for clinical and industrial applications. Here, we identify differences in cell stiffness, expressed as cell elastic modulus (CEM), for hESC versus mesenchymal progenitors, osteoblast-like derivatives, and fibroblasts using atomic force microscopy and data processing algorithms to characterize the stiffness of cell populations. Undifferentiated hESC exhibited a range of CEMs whose median was nearly three-fold lower than those of differentiated cells, information we exploited to develop a label-free separation device based on the principles of tangential flow filtration. To test the device's utility, we segregated hESC mixed with fibroblasts and hESC-mesenchymal progenitors induced to undergo osteogenic differentiation. The device permitted a throughput of 10(6)-10(7) cells per min and up to 50% removal of specific cell types per single pass. The level of enrichment and depletion of soft, pluripotent hESC in the respective channels was found to rise with increasing stiffness of the differentiating cells, suggesting CEM can serve as a major discriminator. Our results demonstrate the principle of a scalable, label-free, solution for separation of heterogeneous cell populations deriving from human pluripotent stem cells. PMID:26858819

  19. A novel in vivo cell-wall labeling approach sheds new light on peptidoglycan synthesis in Escherichia coli

    NARCIS (Netherlands)

    N.K. Olrichs; M.E.G. Aarsman; J. Verheul; C.J. Arnusch; N.I. Martin; M. Hervé; W. Vollmer; B. de Kruijff; E. Breukink; T. den Blaauwen

    2011-01-01

    Peptidoglycan synthesis and turnover in relation to cell growth and division has been studied by using a new labeling method. This method involves the incorporation of fluorescently labeled peptidoglycan precursors into the cell wall by means of the cell-wall recycling pathway. We show that Escheric

  20. Site of fluorescent label modifies interaction of melittin with live cells and model membranes.

    Science.gov (United States)

    Jamasbi, Elaheh; Ciccotosto, Giuseppe D; Tailhades, Julien; Robins-Browne, Roy M; Ugalde, Cathryn L; Sharples, Robyn A; Patil, Nitin; Wade, John D; Hossain, Mohammed Akhter; Separovic, Frances

    2015-10-01

    The mechanism of membrane disruption by melittin (MLT) of giant unilamellar vesicles (GUVs) and live cells was studied using fluorescence microscopy and two fluorescent synthetic analogues of MLT. The N-terminus of one of these was acylated with thiopropionic acid to enable labeling with maleimido-AlexaFluor 430 to study the interaction of MLT with live cells. It was compared with a second analogue labeled at P14C. The results indicated that the fluorescent peptides adhered to the membrane bilayer of phosphatidylcholine GUVs and inserted into the plasma membrane of HeLa cells. Fluorescence and light microscopy revealed changes in cell morphology after exposure to MLT peptides and showed bleb formation in the plasma membrane of HeLa cells. However, the membrane disruptive effect was dependent upon the location of the fluorescent label on the peptide and was greater when MLT was labeled at the N-terminus. Proline at position 14 appeared to be important for antimicrobial activity, hemolysis and cytotoxicity, but not essential for cell membrane disruption. PMID:26051124

  1. Label-free imaging to study phenotypic behavioural traits of cells in complex co-cultures

    Science.gov (United States)

    Suman, Rakesh; Smith, Gabrielle; Hazel, Kathryn E. A.; Kasprowicz, Richard; Coles, Mark; O'Toole, Peter; Chawla, Sangeeta

    2016-02-01

    Time-lapse imaging is a fundamental tool for studying cellular behaviours, however studies of primary cells in complex co-culture environments often requires fluorescent labelling and significant light exposure that can perturb their natural function over time. Here, we describe ptychographic phase imaging that permits prolonged label-free time-lapse imaging of microglia in the presence of neurons and astrocytes, which better resembles in vivo microenvironments. We demonstrate the use of ptychography as an assay to study the phenotypic behaviour of microglial cells in primary neuronal co-cultures through the addition of cyclosporine A, a potent immune-modulator.

  2. A simple method for the measurement of labelled compound incorporation into cells in culture.

    Science.gov (United States)

    Suplisson, A; Boissel, J P

    1976-02-10

    A simple method for the measurement of labelled compound incorporation into cells in layer culture was developed. Compared to other methods it proves to spare time and to be more sensitive owing to the fact that cells are not detached from the culture vials until the end of the manipulation as these are dissolved in the scintillation medium together with the cells just before counting.

  3. Cationic Gd-DTPA liposomes for highly efficient labeling of mesenchymal stem cells and cell tracking with MRI

    NARCIS (Netherlands)

    J. Guenoun (Jamal); G.A. Koning (Gerben); R.Q. Doeswijk (Ronald); L. Bosman (Lizanne); P.A. Wielopolski (Piotr); G.P. Krestin (Gabriel); M.R. Bernsen (Monique)

    2012-01-01

    textabstractIn the current study cell labeling was performed with water-soluble gadolinium (Gd)-DTPA containing liposomes, to allow for cell tracking by MRI. Liposomes were used to assure a highly concentrated intracellular build up of Gd, aiming to overcome the relatively low MRI sensitivity of Gd

  4. Safety and radiation risks in the labelling of blood cells

    International Nuclear Information System (INIS)

    Risk in the management of radioactive material and biological exposition to infectious agents. Protocols and normative to observe GOOD RADIOPHARMACY Practices. Main infectious agents that may be transmitted during preparation of a blood cell radiopharmaceutical. Problems of contamination

  5. Superparamagnetic iron oxide nanoparticles label human bone marrow and umbilical cord mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Ma Yan; Zhang De-qing; Chen Le; Wang Jian; Zhang Xue; Hou Yan; Bi Xiao-juan; Yang Rong; Hu An-hua

    2012-01-01

      BACKGROUND: Nowadays, it is becoming more and more important to optimize safety of human derived cel s, label cel s efficiently and track cel s after cel s transplantation both in basic research and clinic application. OBJECTIVE: To compare the cel viability, labeling efficiency and imaging effect of the T2* weight image (WI) magnetic resonance (MR) between the human bone marrow and umbilical cord derived mesenchymal stem cel s labeled with the superparamaganetic iron oxide nanoparticles, as wel as to optimize their treatment efficiency. METHODS: The third generation of human bone marrow and umbilical cord derived mesenchymal stem cel s were cultured, and labeled with 5-30 mg/L Feridex Ⅳ and protamine sulfate. RESULTS AND CONCLUSION: The viability of human bone marrow mesenchymal stromal cel s was similar with human umbilical cord derived mesenchymal stem cel s (P >0.05). There was no significant difference of labeling rate between the bone marrow msenchymal stem cel s labeled with 5-30 mg/L Feridex Ⅳ(P >0.05); while there was significant difference of labeling rate between the umbilical cord derived mesenchymal stem cel s labeled with 5 mg/L Feridex Ⅳ and 20 and 30 mg/L Feridex Ⅳ(P <0.05); the positive labeling rate of umbilical cord derived mesenchymal stem cel s was lower than that of bone marrow msenchymal stem cel s after labeled with 10 mg/L FeridexⅣ(P <0.05). When two sources of cel s were labeled with Feridex Ⅳ more than 2 mg/L, the iron oxide particles were found in the cel suspension and could not be removed by elution and filtration. The signal intensity from 3.0T MR GRE T2*WI scan was decreased with the increasing of Feridex Ⅳ concentration in both cel types. It is safe and effective to label the two tissue-derived mesenchymal stem cel s with 10 mg/L Feridex Ⅳ-protamine sulfate complex, and can be observed with T2*WI MR.

  6. A novel in vivo cell-wall labeling approach sheds new light on peptidoglycan synthesis in Escherichia coli.

    Science.gov (United States)

    Olrichs, Nick K; Aarsman, Mirjam E G; Verheul, Jolanda; Arnusch, Christopher J; Martin, Nathaniel I; Hervé, Mireille; Vollmer, Waldemar; de Kruijff, Ben; Breukink, Eefjan; den Blaauwen, Tanneke

    2011-05-01

    Peptidoglycan synthesis and turnover in relation to cell growth and division has been studied by using a new labeling method. This method involves the incorporation of fluorescently labeled peptidoglycan precursors into the cell wall by means of the cell-wall recycling pathway. We show that Escherichia coli is able to import exogenous added murein tripeptide labeled with N-7-nitro-2,1,3-benzoxadiazol-4-yl (AeK-NBD) into the cytoplasm where it enters the peptidoglycan biosynthesis route, resulting in fluorescent labels specifically located in the cell wall. When wild-type cells were grown in the presence of the fluorescent peptide, peptidoglycan was uniformly labeled in cells undergoing elongation. Cells in the process of division displayed a lack of labeled peptidoglycan at mid-cell. Analysis of labeling patterns in cell division mutants showed that the occurrence of unlabeled peptidoglycan is dependent on the presence of FtsZ, but independent of FtsQ and FtsI. Accumulation of fluorescence at the division sites of a triple amidase mutant (ΔamiABC) revealed that AeK-NBD is incorporated into septal peptidoglycan. AmiC was shown to be involved in the rapid removal of labeled peptidoglycan side chains at division sites in wild-type cells. Because septal localization of AmiC is dependent on FtsQ and FtsI, this points to the presence of another peptidoglycan hydrolase activity directly dependent on FtsZ. PMID:21472954

  7. Label-free measuring and mapping of binding kinetics of membrane proteins in single living cells

    Science.gov (United States)

    Wang, Wei; Yang, Yunze; Wang, Shaopeng; Nagaraj, Vinay J.; Liu, Qiang; Wu, Jie; Tao, Nongjian

    2012-10-01

    Membrane proteins mediate a variety of cellular responses to extracellular signals. Although membrane proteins are studied intensively for their values as disease biomarkers and therapeutic targets, in situ investigation of the binding kinetics of membrane proteins with their ligands has been a challenge. Traditional approaches isolate membrane proteins and then study them ex situ, which does not reflect accurately their native structures and functions. We present a label-free plasmonic microscopy method to map the local binding kinetics of membrane proteins in their native environment. This analytical method can perform simultaneous plasmonic and fluorescence imaging, and thus make it possible to combine the strengths of both label-based and label-free techniques in one system. Using this method, we determined the distribution of membrane proteins on the surface of single cells and the local binding kinetic constants of different membrane proteins. Furthermore, we studied the polarization of the membrane proteins on the cell surface during chemotaxis.

  8. Differential phospholipid-labeling suggests two subtypes of phospholipase D in rat Leydig cells

    DEFF Research Database (Denmark)

    Lauritzen, L.; Hansen, Harald S.

    1995-01-01

    The aim of the present study was to compare the transphosphatidylation activity of phospholipase D (PLD) under different substrate labeling conditions, in order to investigate whether PLD in rat Leydig cells exhibited any substrate preferences for the alkyl- or acyl-form of phosphatidylcholine (Ptd......Cho). The [H] phosphatidylethanol formation in response to 4ß-phorbol 12-myristate 13-acetate (PMA), sphingosine, or Ca-ionophore A23187, was lower when Leydig cells were labeled with 1-O-[H]alkyl lysoPtdCho compared with the responses when [H]myristic acid was employed. In contrast, the results...... for the receptor agonists (vasopressin, bradykinin, and lysophosphatidic acid), using the two labels, showed mole consistency. Thus, the PLD-activity induced by PMA, sphingosine, or A23187 has a more selective substrate range (i.e. mainly acyl-linked PtdCho) than the PLD-activity stimulated via a receptor. Our...

  9. Targeting single neuronal networks for gene expression and cell labeling in vivo.

    Science.gov (United States)

    Marshel, James H; Mori, Takuma; Nielsen, Kristina J; Callaway, Edward M

    2010-08-26

    To understand fine-scale structure and function of single mammalian neuronal networks, we developed and validated a strategy to genetically target and trace monosynaptic inputs to a single neuron in vitro and in vivo. The strategy independently targets a neuron and its presynaptic network for specific gene expression and fine-scale labeling, using single-cell electroporation of DNA to target infection and monosynaptic retrograde spread of a genetically modifiable rabies virus. The technique is highly reliable, with transsynaptic labeling occurring in every electroporated neuron infected by the virus. Targeting single neocortical neuronal networks in vivo, we found clusters of both spiny and aspiny neurons surrounding the electroporated neuron in each case, in addition to intricately labeled distal cortical and subcortical inputs. This technique, broadly applicable for probing and manipulating single neuronal networks with single-cell resolution in vivo, may help shed new light on fundamental mechanisms underlying circuit development and information processing by neuronal networks throughout the brain.

  10. Discrimination of bromodeoxyuridine labelled and unlabelled mitotic cells in flow cytometric bromodeoxyuridine/DNA analysis

    DEFF Research Database (Denmark)

    Jensen, P O; Larsen, J K; Christensen, I J;

    1994-01-01

    Bromodeoxyuridine (BrdUrd) labelled and unlabelled mitotic cells, respectively, can be discriminated from interphase cells using a new method, based on immunocytochemical staining of BrdUrd and flow cytometric four-parameter analysis of DNA content, BrdUrd incorporation, and forward and orthogonal...... light scatter. The method was optimized using the human leukemia cell lines HL-60 and K-562. Samples of 10(5) ethanol-fixed cells were treated with pepsin/HCl and stained as a nuclear suspension with anti-BrdUrd antibody, FITC-conjugated secondary antibody, and propidium iodide. Labelled mitoses could...... fluorescence distribution. This interpretation was supported by experiments using mitotic arrest, fluorescence activated cell sorting and microscopy, and comparison with an alternative flow cytometric method for discrimination of mitoses....

  11. SKOV-3 cell imaging by paramagnetic particles labeled with hairpin cell-penetrating peptides

    Institute of Scientific and Technical Information of China (English)

    ZHAI Xiao-hui; LIU Min; GUO Xiao-juan; WANG Si-cen; ZHANG Hong-xia; GUO You-min

    2011-01-01

    Background The hairpin cell-penetrating peptides (hCPPs) demonstrate an interesting characteristic of conditioned activation by molecules. We hypothesized that hCPPs have the potential to selectively deliver a paramagnetic gadolinium probe into the matrix metalloproteinase 2 (MMP-2) positive human ovary adenocarcinoma cell lines,SKOV-3.Methods hCPPs were synthesized and labeled with 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid gadolinium (Ⅲ) (Gd-DOTA) and fluorescein isothiocyanate (FITC) by f-moc strategy using a standard solid phase peptide synthesis protocol. MMP-2 expression and activity were demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) and zymography. Internalization and location of hCPPs in SKOV-3 cells were observed by fluorescein imaging and flow cytometery. Selective delivery of Gd-DOTA in SKOV-3 cells was observed by magnetic resonance imaging (MRI) and transmission electron microscopy (TEM).Results The uptake of hCPPs by SKOV-3 cells depended on the activity of MMP-2. T1WI signals of SKOV-3 cells treated with Gd-DOTA-hCPPs suggested the uptake of Gd-DOTA-hCPPs increased in a time- (r=0.990, P <0.01) and concentration-dependent manner (r=0.964, P <0.001), but was inhibited by a MMP-2 inhibitor. Electron-dense particles observed in the cytoplasm and nucleus by transmission electron microscopy proved the intracellular penetration of gadolinium.Conclusions hCPPs can be used as an effective vector for an MRI molecular probe to assess the activity of MMP-2.

  12. Nestin Reporter Transgene Labels Multiple Central Nervous System Precursor Cells

    Directory of Open Access Journals (Sweden)

    Avery S. Walker

    2010-01-01

    Full Text Available Embryonic neuroepithelia and adult subventricular zone (SVZ stem and progenitor cells express nestin. We characterized a transgenic line that expresses enhanced green fluorescent protein (eGFP specified to neural tissue by the second intronic enhancer of the nestin promoter that had several novel features. During embryogenesis, the dorsal telencephalon contained many and the ventral telencephalon few eGFP+ cells. eGFP+ cells were found in postnatal and adult neurogenic regions. eGFP+ cells in the SVZ expressed multiple phenotype markers, glial fibrillary acidic protein, Dlx, and neuroblast-specific molecules suggesting the transgene is expressed through the lineage. eGFP+ cell numbers increased in the SVZ after cortical injury, suggesting this line will be useful in probing postinjury neurogenesis. In non-neurogenic regions, eGFP was strongly expressed in oligodendrocyte progenitors, but not in astrocytes, even when they were reactive. This eGFP+ mouse will facilitate studies of proliferative neuroepithelia and adult neurogenesis, as well as of parenchymal oligodendrocytes.

  13. Immunospecific red cell binding of iodine /sup 125/-labeled immunoglobulin G erythrocyte autoantibodies

    Energy Technology Data Exchange (ETDEWEB)

    Masouredis, S.P.; Branks, M.J.; Garratty, G.; Victoria, E.J.

    1987-09-01

    The primary interaction of autoantibodies with red cells has been studied by using labeled autoantibodies. Immunoglobulin G red cell autoantibodies obtained from IgG antiglobulin-positive normal blood donors were labeled with radioactive iodine and compared with alloanti-D with respect to their properties and binding behavior. Iodine /sup 125/-labeled IgG autoantibody migrated as a single homogeneous peak with the same relative mobility as human IgG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric focusing pattern of labeled autoantibodies varied from donor to donor but was similar to that of alloanti-D, consisting of multiple IgG populations with isoelectric points in the neutral to alkaline range. /sup 125/I-autoantibody bound to all human red cells of common Rh phenotypes. Evidence for immunospecific antibody binding of the labeled autoantibody was based on variation in equilibrium binding to nonhuman and human red cells of common and rare phenotypes, enhanced binding after red cell protease modification, antiglobulin reactivity of cell-bound IgG comparable to that of cell-bound anti-D, and saturation binding in autoantibody excess. Scatchard analysis of two /sup 125/I-autoantibody preparations yielded site numbers of 41,500 and 53,300 with equilibrium constants of 3.7 and 2.1 X 10(8) L X mol-1. Dog, rabbit, rhesus monkey, and baboon red cells were antigen(s) negative by quantitative adsorption studies adsorbing less than 3% of the labeled autoantibody. Reduced ability of rare human D--red blood cells to adsorb the autoantibody and identification of donor autoantibodies that bind to Rh null red blood cells indicated that eluates contained multiple antibody populations of complex specificities in contrast to anti-D, which consists of a monospecific antibody population. Another difference is that less than 70% of the autoantibody IgG was adsorbed by maximum binding red blood cells as compared with greater than 85% for alloanti-D.

  14. Bioprinting for stem cell research

    OpenAIRE

    Tasoglu, Savas; Demirci, Utkan

    2012-01-01

    Recently, there has been a growing interest to apply bioprinting techniques to stem cell research. Several bioprinting methods have been developed utilizing acoustics, piezoelectricity, and lasers to deposit living cells onto receiving substrates. Using these technologies, spatially defined gradients of immobilized proteins can be engineered to direct stem cell differentiation into multiple subpopulations of different lineages. Stem cells can also be patterned in a high-throughput manner onto...

  15. Noninvasive and label-free determination of virus infected cells by Raman spectroscopy

    Science.gov (United States)

    Moor, Kamila; Ohtani, Kiyoshi; Myrzakozha, Diyas; Zhanserkenova, Orik; Andriana, Bibin. B.; Sato, Hidetoshi

    2014-06-01

    The present study demonstrates that Raman spectroscopy is a powerful tool for the detection of virus-infected cells. Adenovirus infection of human embryonic kidney 293 cells was successfully detected at 12, 24, and 48 h after initiating the infection. The score plot of principal component analysis discriminated the spectra of the infected cells from those of the control cells. The viral infection was confirmed by the conventional immunostaining method performed 24 h after the infection. The newly developed method provides a fast and label-free means for the detection of virus-infected cells.

  16. Use of indium-111-labeled white blood cells in the diagnosis of diabetic foot infections

    Energy Technology Data Exchange (ETDEWEB)

    Zeiger, L.S.; Fox, I.M.

    1990-01-01

    The diagnosis of bone infection in the patient with nonvirgin bone is a diagnostic dilemma. This is especially true in the diabetic patient with a soft tissue infection and an underlying osteoarthropathy. The authors present a retrospective study using the new scintigraphic technique of indium-111-labeled white blood cells as a method of attempting to solve this diagnostic dilemma.

  17. Clinical impact of ki-67 labeling index in non-small cell lung cancer

    DEFF Research Database (Denmark)

    Jakobsen, Jan Nyrop; Sørensen, Jens Benn

    2013-01-01

    The ki-67 index is a marker of proliferation in malignant tumors. Studies from the period 2000 to 2012 on the prognostic and predictive value of ki-67 labeling index (LI) in non-small cell cancer (NSCLC) are reviewed. Twenty-eight studies reported on the prognostic value of ki-67 index with various...

  18. Selective labelling of cell-surface proteins using CyDye DIGE Fluor minimal dyes.

    Science.gov (United States)

    Hagner-McWhirter, Asa; Winkvist, Maria; Bourin, Stephanie; Marouga, Rita

    2008-01-01

    Surface proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of almost all intracellular signaling. Moreover, they play an important role in environmental adaptation and drug treatment, and are often involved in disease pathogenesis and pathology (1). Protein-protein interactions are intrinsic to signaling pathways, and to gain more insight in these complex biological processes, sensitive and reliable methods are needed for studying cell surface proteins. Two-dimensional (2-D) electrophoresis is used extensively for detection of biomarkers and other targets in complex protein samples to study differential changes. Cell surface proteins, partly due to their low abundance (1 2% of cellular proteins), are difficult to detect in a 2-D gel without fractionation or some other type of enrichment. They are also often poorly represented in 2-D gels due to their hydrophobic nature and high molecular weight (2). In this study, we present a new protocol for intact cells using CyDye DIGE Fluor minimal dyes for specific labeling and detection of this important group of proteins. The results showed specific labeling of a large number of cell surface proteins with minimal labeling of intracellular proteins. This protocol is rapid, simple to use, and all three CyDye DIGE Fluor minimal dyes (Cy 2, Cy 3 and Cy 5) can be used to label cell-surface proteins. These features allow for multiplexing using the 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) with Ettan DIGE technology and analysis of protein expression changes using DeCyder 2-D Differential Analysis Software. The level of cell-surface proteins was followed during serum starvation of CHO cells for various lengths of time (see Table 1). Small changes in abundance were detected with high accuracy, and results are supported by defined statistical methods.

  19. Magnetic resonance imaging of single co-labeled mesenchymal stromal cells after intracardial injection in mice

    Energy Technology Data Exchange (ETDEWEB)

    Salamon, J.; Adam, G.; Peldschus, K. [University Medical Center Hamburg-Eppendorf, Hamburg (Germany). Dept. of Diagnostic and Interventional Radiology; Wicklein, D.; Schumacher, U. [University Medical Center Hamburg-Eppendorf, Hamburg (Germany). Inst. of Anatomy II: Experimental Morphology; Didie, M. [Goettingen Univ. (Germany). Inst. of Pharmacology; Lange, C. [University Medical Center Hamburg-Eppendorf, Hamburg (Germany). Dept. of Bone Marrow Transplantation

    2014-04-15

    Purpose: The aim of this study was to establish co-labeling of mesenchymal stromal cells (MSC) for the detection of single MSC in-vivo by MRI and histological validation. Materials and Methods: Mouse MSC were co-labeled with fluorescent iron oxide micro-particles and carboxyfluorescein succinimidyl ester (CFSE). The cellular iron content was determined by atomic absorption spectrometry. Cell proliferation and expression of characteristic surface markers were determined by flow cytometry. The chondrogenic differentiation capacity was assessed. Different amounts of cells (n1 = 5000, n2 = 15 000, n3 = 50 000) were injected into the left heart ventricle of 12 mice. The animals underwent sequential MRI on a clinical 3.0T scanner (Intera, Philips Medical Systems, Best, The Netherlands). For histological validation cryosections were examined by fluorescent microscopy. Results: Magnetic and fluorescent labeling of MSC was established (mean cellular iron content 23.6 ± 3 pg). Flow cytometry showed similar cell proliferation and receptor expression of labeled and unlabeled MSC. Chondrogenic differentiation of labeled MSC was verified. After cell injection MRI revealed multiple signal voids in the brain and fewer signal voids in the kidneys. In the brain, an average of 4.6 ± 1.2 (n1), 9.0 ± 3.6 (n2) and 25.0 ± 1.0 (n3) signal voids were detected per MRI slice. An average of 8.7 ± 3.1 (n1), 22.0 ± 6.1 (n2) and 89.8 ± 6.5 (n3) labeled cells per corresponding stack of adjacent cryosections could be detected in the brain. Statistical correlation of the numbers of MRI signal voids in the brain and single MSC found by histology revealed a correlation coefficient of r = 0.91. Conclusion: The study demonstrates efficient magnetic and fluorescent co-labeling of MSC and their detection on a single cell level in mice by in-vivo MRI and histology. The described techniques may broaden the methods for in-vivo tracking of MSC. (orig.)

  20. Preparation of iron oxide-entrapped chitosan nanoparticles for stem cell labeling.

    Science.gov (United States)

    Chaleawlert-Umpon, Saowaluk; Mayen, Varissaporn; Manotham, Krissanapong; Pimpha, Nuttaporn

    2010-01-01

    This study intended to prepare iron oxide nanoparticle-entrapped chitosan (CS) nanoparticles for stem cell labeling. The nanoparticles were synthesized by polymerizing iron oxide nanoparticle-associated methacrylic acid monomer in the presence of CS. TEM revealed that the well-defined iron oxide nanoparticles were successfully encapsulated inside the CS nanoparticles. The effect of CS at different [NH(2)]/[COOH] molar ratios on particle size, surface charge, thermal stability and magnetic properties was determined systematically. Internalization and localization of the coated nanoparticles were evaluated by atomic absorption spectrometry and confocal laser scanning microscopy. The Kusa O cell line was chosen as a stem cell model. Interestingly, the uptake of iron oxide-entrapped CS nanoparticles was remarkably enhanced under magnetization and the nanoparticles were mostly located inside cellular compartments. It can be concluded that the iron oxide-entrapped CS nanoparticles have a strong potential for stem cell labeling. PMID:20537238

  1. Serial in vivo imaging of the porcine heart after percutaneous, intramyocardially injected 111In-labeled human mesenchymal stromal cells

    DEFF Research Database (Denmark)

    Lyngbaek, Stig; Ripa, Rasmus S; Haack-Sørensen, Mandana;

    2010-01-01

    This pilot trial aimed to investigate the utilization of (111)In-labeling of mesenchymal stromal cells (MSC) for in vivo tracking after intramyocardial transplantation in a xenotransplantation model with gender mismatched cells. Human male MSC were expanded ex vivo and labeled with (111)In...

  2. Serial in vivo imaging of the porcine heart after percutaneous, intramyocardially injected (111)In-labeled human mesenchymal stromal cells

    DEFF Research Database (Denmark)

    Lyngbæk, Stig; Ripa, Rasmus Sejersten; Haack-Sørensen, Mandana;

    2009-01-01

    This pilot trial aimed to investigate the utilization of (111)In-labeling of mesenchymal stromal cells (MSC) for in vivo tracking after intramyocardial transplantation in a xenotransplantation model with gender mismatched cells. Human male MSC were expanded ex vivo and labeled with (111)In...

  3. Novel positively charged nanoparticle labeling for in vivo imaging of adipose tissue-derived stem cells.

    Directory of Open Access Journals (Sweden)

    Hiroshi Yukawa

    Full Text Available Stem cell transplantation has been expected to have various applications for regenerative medicine. However, in order to detect and trace the transplanted stem cells in the body, non-invasive and widely clinically available cell imaging technologies are required. In this paper, we focused on magnetic resonance (MR imaging technology, and investigated whether the trimethylamino dextran-coated magnetic iron oxide nanoparticle -03 (TMADM-03, which was newly developed by our group, could be used for labeling adipose tissue-derived stem cells (ASCs as a contrast agent. No cytotoxicity was observed in ASCs transduced with less than 100 µg-Fe/mL of TMADM-03 after a one hour transduction time. The transduction efficiency of TMADM-03 into ASCs was about four-fold more efficient than that of the alkali-treated dextran-coated magnetic iron oxide nanoparticle (ATDM, which is a major component of commercially available contrast agents such as ferucarbotran (Resovist, and the level of labeling was maintained for at least two weeks. In addition, the differentiation ability of ASCs labeled with TMADM-03 and their ability to produce cytokines such as hepatocyte growth factor (HGF, vascular endothelial growth factor (VEGF and prostaglandin E2 (PGE2, were confirmed to be maintained. The ASCs labeled with TMADM-03 were transplanted into the left kidney capsule of a mouse. The labeled ASCs could be imaged with good contrast using a 1T MR imaging system. These data suggest that TMADM-03 can therefore be utilized as a contrast agent for the MR imaging of stem cells.

  4. Radionuclide imaging of primary renal-cell carcinoma by I-131-labeled antitumor antibody

    International Nuclear Information System (INIS)

    A goat antibody against human renal-cell carcinoma reacted on immunofluorescence with renal-cell carcinomas from 20 patients, but not with normal adult human tissues, including kidney. After i.v. administration the I-131-linked antibody showed preferential tumor localization in six of seven patients with primary renal carcinoma. Labeled antitumor antibodies may have the specificity for tumor imaging that current radiopharmaceuticals lack

  5. Label-free haemogram using wavelength modulated Raman spectroscopy for identifying immune-cell subset

    Science.gov (United States)

    Ashok, Praveen C.; Praveen, Bavishna B.; Campbell, Elaine C.; Dholakia, Kishan; Powis, Simon J.

    2014-03-01

    Leucocytes in the blood of mammals form a powerful protective system against a wide range of dangerous pathogens. There are several types of immune cells that has specific role in the whole immune system. The number and type of immune cells alter in the disease state and identifying the type of immune cell provides information about a person's state of health. There are several immune cell subsets that are essentially morphologically identical and require external labeling to enable discrimination. Here we demonstrate the feasibility of using Wavelength Modulated Raman Spectroscopy (WMRS) with suitable machine learning algorithms as a label-free method to distinguish between different closely lying immune cell subset. Principal Component Analysis (PCA) was performed on WMRS data from single cells, obtained using confocal Raman microscopy for feature reduction, followed by Support Vector Machine (SVM) for binary discrimination of various cell subset, which yielded an accuracy >85%. The method was successful in discriminating between untouched and unfixed purified populations of CD4+CD3+ and CD8+CD3+ T lymphocyte subsets, and CD56+CD3- natural killer cells with a high degree of specificity. It was also proved sensitive enough to identify unique Raman signatures that allow clear discrimination between dendritic cell subsets, comprising CD303+CD45+ plasmacytoid and CD1c+CD141+ myeloid dendritic cells. The results of this study clearly show that WMRS is highly sensitive and can distinguish between cell types that are morphologically identical.

  6. Label-free measurements on cell apoptosis using a terahertz metamaterial-based biosensor

    Science.gov (United States)

    Zhang, Caihong; Liang, Lanju; Ding, Liang; Jin, Biaobing; Hou, Yayi; Li, Chun; Jiang, Ling; Liu, Weiwei; Hu, Wei; Lu, Yanqing; Kang, Lin; Xu, Weiwei; Chen, Jian; Wu, Peiheng

    2016-06-01

    Label-free, real-time, and in-situ measurement on cell apoptosis is highly desirable in cell biology. We propose here a design of terahertz (THz) metamaterial-based biosensor for meeting this requirement. This metamaterial consists of a planar array of five concentric subwavelength gold ring resonators on a 10 μm-thick polyimide substrate, which can sense the change of dielectric environment above the metamaterial. We employ this sensor to an oral cancer cell (SCC4) with and without cisplatin, a chemotherapy drug for cancer treatment, and find a linear relation between cell apoptosis measured by Flow Cytometry and the relative change of resonant frequencies of the metamaterial measured by THz time-domain spectroscopy. This implies that we can determine the cell apoptosis in a label-free manner. We believe that this metamaterial-based biosensor can be developed into a cheap, label-free, real-time, and in-situ detection tool, which is of significant impact on the study of cell biology.

  7. Monitoring Astrocytic Proteome Dynamics by Cell Type-Specific Protein Labeling.

    Directory of Open Access Journals (Sweden)

    Anke Müller

    Full Text Available The ability of the nervous system to undergo long-term plasticity is based on changes in cellular and synaptic proteomes. While many studies have explored dynamic alterations in neuronal proteomes during plasticity, there has been less attention paid to the astrocytic counterpart. Indeed, progress in identifying cell type-specific proteomes is limited owing to technical difficulties. Here, we present a cell type-specific metabolic tagging technique for a mammalian coculture model based on the bioorthogonal amino acid azidonorleucine and the mutated Mus musculus methionyl-tRNA synthetaseL274G enabling azidonorleucine introduction into de novo synthesized proteins. Azidonorleucine incorporation resulted in cell type-specific protein labeling and retained neuronal or astrocytic cell viability. Furthermore, we were able to label astrocytic de novo synthesized proteins and identified both Connexin-43 and 60S ribosomal protein L10a upregulated upon treatment with Brain-derived neurotrophic factor in astrocytes of a neuron-glia coculture. Taken together, we demonstrate the successful dissociation of astrocytic from neuronal proteomes by cell type-specific metabolic labeling offering new possibilities for the analyses of cell type-specific proteome dynamics.

  8. Labelling techniques of biomolecules for targeted radiotherapy. Final report of a co-ordinated research project 1998-2002

    International Nuclear Information System (INIS)

    Malignant tumour disease accounts for approximately one third of deaths worldwide. Gastrointestinal adenocarcinomas, prostate and breast cancers are among the most frequently appearing tumours. Radiotherapy is an essential mode of treatment of all cancer patients either alone or in conjunction with other modalities like surgery and chemotherapy. In most cases radiotherapy is given using external radiation sources. It is also possible to administer radiotherapy by specifically localizing radioisotopes emitting particulate radiation in the tumour tissue. This targeted therapy has proved to have several advantages over external beam therapy, notably the possibility of selectively delivering higher radiation doses to the targeted tumour cells and treating multiple metastases. Procedures for therapy of thyroid carcinoma and hyper-thyroidism using radioiodine (131I) introduced about five decades ago, have stood the test of time and are still widely used the world over. In addition to the therapeutic nuclides of the first generation 131I, 89Sr, 32P, 90Y, etc., which are still widely utilized and accepted by the medical community, many other beta emitting radionuclides with relatively short half-lives such as 153Sm, 186Re, 188Re, 166Ho, 165Dy, etc. have also been recently made available for therapy and used with promising good results. In spite of the potential of targeted radiotherapy to treat a wide range of malignant conditions, routine clinical use is mostly confined to therapy of thyroid carcinoma, hyperthyroidism, metastatic bone pain and synovectomy. In most of the cases, the limitation is obviously not the availability of suitable radionuclides but rather the lack of suitable carrier molecules that would adequately concentrate these radionuclides in target tissues of interest. Based on the above considerations, the scope of the Co-ordinated Research Project (CRP) has focused on the synthesis of the required BFCAs for MoAbs and peptide labelling, development and

  9. Label-free detection of anticancer drug paclitaxel in living cells by confocal Raman microscopy

    Science.gov (United States)

    Salehi, H.; Derely, L.; Vegh, A.-G.; Durand, J.-C.; Gergely, C.; Larroque, C.; Fauroux, M.-A.; Cuisinier, F. J. G.

    2013-03-01

    Confocal Raman microscopy, a non-invasive, label-free, and high spatial resolution imaging technique is employed to trace the anticancer drug paclitaxel in living Michigan Cancer Foundation-7 (MCF-7) cells. The Raman images were treated by K-mean cluster analysis to detect the drug in cells. Distribution of paclitaxel in cells is verified by calculating the correlation coefficient between the reference spectrum of the drug and the whole Raman image spectra. A time dependent gradual diffusion of paclitaxel all over the cell is observed suggesting a complementary picture of the pharmaceutical action of this drug based on rapid binding of free tubulin to crystallized paclitaxel.

  10. Quantitative Label-Free Cell Proliferation Tracking with a Versatile Electrochemical Impedance Detection Platform

    DEFF Research Database (Denmark)

    Caviglia, Claudia; Carminati, M; Heiskanen, Arto;

    2012-01-01

    optimal detection strategies. Electrochemical Impedance Spectroscopy (EIS) has been used to monitor and compare adhesion of different cell lines. HeLa cells and 3T3 fibroblasts have been cultured for 12 hours on interdigitated electrode arrays integrated into a tailor-made cell culture platform. Both......Since the use of impedance measurements for label-free monitoring of cells has become widespread but still the choice of sensing configuration is not unique though crucial for a quantitative interpretation of data, we demonstrate the application of a novel custom multipotentiostat platform to study...

  11. MR tracking of SPIO-labeled mesenchymal stem cells in rats with liver fibrosis could not monitor the cells accurately.

    Science.gov (United States)

    Zhou, Bin; Li, Dan; Qian, Jiesheng; Li, Zhengran; Pang, Pengfei; Shan, Hong

    2015-01-01

    Our previous study showed that in vivo magnetic resonance (MR) imaging is effective in tracking superparamagnetic iron oxide (SPIO)-labeled bone marrow mesenchymal stem cells (BMSCs) in rats with liver fibrosis. SPIO-labeling-induced signal reduction on MR images was completely reversed within 15 days after transplantation. It is still unclear whether the signal changes in MR imaging could reflect the number of transplanted cells in the liver. In the present study, BMSCs of male rats were doubly labeled with enhanced green fluorescent protein (EGFP) and SPIO and injected intravascularly into female rats with liver fibrosis. At different time points after injection, MR imaging was performed. The distribution of SPIO particles and EGFP-positive cells was determined by Prussian blue staining and EGFP immunohistochemistry, respectively. The distribution of transplanted BMSCs in various organs was assessed by detection of the SRY gene using real-time quantitative PCR. At 15 days post transplantation, the numbers of transplanted cells were significantly decreased in the lung, kidney, spleen and muscle, but not liver and heart, in comparison with those at 7 days after transplantation. EGFP staining-positive cells were observed in the liver intralobular parenchyma, while Prussian blue staining was negative at 42 days after transplantation. Taken together, SPIO particles and EGFP-labeled BMSCs show a different tissue distribution pattern in rats with liver fibrosis after a long-term period of monitoring. SPIO-based MR imaging may not be suitable for long-term tracking of transplanted BMSCs in vivo.

  12. MR tracking of SPIO-labeled mesenchymal stem cells in rats with liver fibrosis could not monitor the cells accurately.

    Science.gov (United States)

    Zhou, Bin; Li, Dan; Qian, Jiesheng; Li, Zhengran; Pang, Pengfei; Shan, Hong

    2015-01-01

    Our previous study showed that in vivo magnetic resonance (MR) imaging is effective in tracking superparamagnetic iron oxide (SPIO)-labeled bone marrow mesenchymal stem cells (BMSCs) in rats with liver fibrosis. SPIO-labeling-induced signal reduction on MR images was completely reversed within 15 days after transplantation. It is still unclear whether the signal changes in MR imaging could reflect the number of transplanted cells in the liver. In the present study, BMSCs of male rats were doubly labeled with enhanced green fluorescent protein (EGFP) and SPIO and injected intravascularly into female rats with liver fibrosis. At different time points after injection, MR imaging was performed. The distribution of SPIO particles and EGFP-positive cells was determined by Prussian blue staining and EGFP immunohistochemistry, respectively. The distribution of transplanted BMSCs in various organs was assessed by detection of the SRY gene using real-time quantitative PCR. At 15 days post transplantation, the numbers of transplanted cells were significantly decreased in the lung, kidney, spleen and muscle, but not liver and heart, in comparison with those at 7 days after transplantation. EGFP staining-positive cells were observed in the liver intralobular parenchyma, while Prussian blue staining was negative at 42 days after transplantation. Taken together, SPIO particles and EGFP-labeled BMSCs show a different tissue distribution pattern in rats with liver fibrosis after a long-term period of monitoring. SPIO-based MR imaging may not be suitable for long-term tracking of transplanted BMSCs in vivo. PMID:26153152

  13. The use of dopamine-hyaluronate associate-coated maghemite nanoparticles to label cells

    Directory of Open Access Journals (Sweden)

    Babic M

    2012-03-01

    Full Text Available Michal Babic1,2, Daniel Horak1,2, Pavla Jendelova2,3, Vit Herynek2,4, Vladimir Proks1,2, Vaclav Vanecek2,3, Petr Lesny2,3, Eva Sykova2,31Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq 2, 2Center for Cell Therapy and Tissue Repair, Charles University, V Uvalu 84, 3Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Videnska 1083, 4Institute for Clinical and Experimental Medicine, Videnska 1958, Prague, Czech RepublicAbstract: Sodium hyaluronate (HA was associated with dopamine (DPA and introduced as a coating for maghemite (γ-Fe2O3 nanoparticles obtained by the coprecipitation of iron(II and iron(III chlorides and oxidation with sodium hypochlorite. The effects of the DPA anchorage of HA on the γ-Fe2O3 surface on the physicochemical properties of the resulting colloids were investigated. Nanoparticles coated at three different DPA-HA/γ-Fe2O3 and DPA/HA ratios were chosen for experiments with rat bone marrow mesenchymal stem cells and human chondrocytes. The nanoparticles were internalized into rat bone marrow mesenchymal stem cells via endocytosis as confirmed by Prussian Blue staining. The efficiency of mesenchymal stem cell labeling was analyzed. From among the investigated samples, efficient cell labeling was achieved by using DPA-HA-γ-Fe2O3 nanoparticles with DPA-HA/γ-Fe2O3 = 0.45 (weight/weight and DPA/HA = 0.038 (weight/weight ratios. The particles were used as a contrast agent in magnetic resonance imaging for the labeling and visualization of cells.Keywords: nanoparticles, dopamine, hyaluronate, cell labeling, magnetic

  14. Label-free recognition of drug resistance via impedimetric screening of breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Bilge Eker

    Full Text Available We present a novel study on label-free recognition and distinction of drug resistant breast cancer cells (MCF-7 DOX from their parental cells (MCF-7 WT via impedimetric measurements. Drug resistant cells exhibited significant differences in their dielectric properties compared to wild-type cells, exerting much higher extracellular resistance (Rextra . Immunostaining revealed that MCF-7 DOX cells gained a much denser F-actin network upon acquiring drug resistance indicating that remodeling of actin cytoskeleton is probably the reason behind higher Rextra , providing stronger cell architecture. Moreover, having exposed both cell types to doxorubicin, we were able to distinguish these two phenotypes based on their substantially different drug response. Interestingly, impedimetric measurements identified a concentration-dependent and reversible increase in cell stiffness in the presence of low non-lethal drug doses. Combined with a profound frequency analysis, these findings enabled distinguishing distinct cellular responses during drug exposure within four concentration ranges without using any labeling. Overall, this study highlights the possibility to differentiate drug resistant phenotypes from their parental cells and to assess their drug response by using microelectrodes, offering direct, real-time and noninvasive measurements of cell dependent parameters under drug exposure, hence providing a promising step for personalized medicine applications such as evaluation of the disease progress and optimization of the drug treatment of a patient during chemotherapy.

  15. The effect of the state of differentiation on labeling of epidermal cell surface glycoproteins

    International Nuclear Information System (INIS)

    Epidermal cells were grown in a medium in which the Ca++ concentration controlled the stage of differentiation. Cell surface molecules of differentiated and undifferentiated cells were compared by lactoperoxidase-catalyzed iodination, by the interaction with 125I-lectins, and by the metabolic incorporation of L-(3H)-fucose. Molecular weights of the labeled components were determined by SDS-PAGE and autoradiography. After lactoperoxidase iodination, most of the radioactivity was found in polypeptide bands of 79,000, 65,000 and 56,000 daltons. The 79,000 band is the most intense for undifferentiated cells but disappears as differentiation proceeds. The 56,000 band is present in normal cells at all stages of differentiation but is absent from neoplastic cells. Glycoproteins reacted with 125I-lectins were found at 180,000, 130,000 and 85,000 daltons. The 130,000 band was the most prominent for differentiated cells labeled with wheat germ agglutinin but was essentially absent from the undifferentiated cells. With Ricinus communis agglutinin, this band was weaker for undifferentiated than for differentiated cells but was the most intense for both. After metabolic incorporation of tritiated fucose, radioactive glycoproteins were found at 130,000 and 85,000 daltons, with comparable intensities for differentiated and undifferentiated cells

  16. Fluorophore-conjugated iron oxide nanoparticle labeling and analysis of engrafting human hematopoietic stem cells

    DEFF Research Database (Denmark)

    Maxwell, Dustin J; Bonde, Jesper; Hess, David A;

    2008-01-01

    or in vivo. Transplantation of purified primary human cord blood lineage-depleted and CD34(+) cells into immunodeficient mice allowed detection of labeled human HSC in the recipient bones. Flow cytometry was used to precisely quantitate the cell populations that had sequestered the nanoparticles......The use of nanometer-sized iron oxide particles combined with molecular imaging techniques enables dynamic studies of homing and trafficking of human hematopoietic stem cells (HSC). Identifying clinically applicable strategies for loading nanoparticles into primitive HSC requires strictly defined...... culture conditions to maintain viability without inducing terminal differentiation. In the current study, fluorescent molecules were covalently linked to dextran-coated iron oxide nanoparticles (Feridex) to characterize human HSC labeling to monitor the engraftment process. Conjugating fluorophores...

  17. Self-Assembled Superparamagnetic Iron Oxide Nanoclusters for Universal Cell Labeling and MRI.

    Science.gov (United States)

    Chen, Shuzhen; Zhang, Jun; Jiang, Shengwei; Lin, Gan; Luo, Bing; Yao, Huan; Lin, Yuchun; He, Chengyong; Liu, Gang; Lin, Zhongning

    2016-12-01

    Superparamagnetic iron oxide (SPIO) nanoparticles have been widely used in a variety of biomedical applications, especially as contrast agents for magnetic resonance imaging (MRI) and cell labeling. In this study, SPIO nanoparticles were stabilized with amphiphilic low molecular weight polyethylenimine (PEI) in an aqueous phase to form monodispersed nanocomposites with a controlled clustering structure. The iron-based nanoclusters with a size of 115.3 ± 40.23 nm showed excellent performance on cellular uptake and cell labeling in different types of cells, moreover, which could be tracked by MRI with high sensitivity. The SPIO nanoclusters presented negligible cytotoxicity in various types of cells as detected using MTS, LDH, and flow cytometry assays. Significantly, we found that ferritin protein played an essential role in protecting stress from SPIO nanoclusters. Taken together, the self-assembly of SPIO nanoclusters with good magnetic properties provides a safe and efficient method for universal cell labeling with noninvasive MRI monitoring capability. PMID:27216601

  18. Canine mesenchymal stem cells are effectively labeled with silica nanoparticles and unambiguously visualized in highly autofluorescent tissues

    Directory of Open Access Journals (Sweden)

    Han Sei-Myoung

    2012-08-01

    Full Text Available Abstract Background Development of a method for long-term labeling of cells is critical to elucidate transplanted cell fate and migration as well as the contribution to tissue regeneration. Silica nanoparticles have been recently developed and demonstrated to be biocompatible with a high labeling capacity. Thus, our study was designed to assess the suitability of silica nanoparticles for labeling canine mesenchymal stem cells (MSCs and the fluorescence afficiency in highly autofluorescent tissue. Results We examined the effect of silica nanoparticle labeling on stem cell morphology, viability and differentiation as compared with those of unlabeled control cells. After 4 h of incubation with silica nanoparticles, they were internalized by canine MSCs without a change in the morphology of cells compared with that of control cells. The viability and proliferation of MSCs labeled with silica nanoparticles were evaluated by a WST-1 assay and trypan blue exclusion. No effects on cell viability were observed, and the proliferation of canine MSCs was not inhibited during culture with silica nanoparticles. Furthermore, adipogenic and osteogenic differentiation of silica nanoparticle-labeled canine MSCs was at a similar level compared with that of unlabeled cells, indicating that silica nanoparticle labeling did not alter the differentiation capacity of canine MSCs. Silica nanoparticle-labeled canine MSCs were injected into the kidneys of BALB/c mice after celiotomy, and then the mice were sacrificed after 2 or 3 weeks. The localization of injected MSCs was closely examined in highly autofluorescent renal tissues. Histologically, canine MSCs were uniformly and completely labeled with silica nanoparticles, and were unambiguously imaged in histological sections. Conclusions The results of the current study showed that silica nanoparticles are useful as an effective labeling marker for MSCs, which can elucidate the distribution and fate of transplanted

  19. Stable isotope labeling by amino acids in cell culture (SILAC) and quantitative comparison of the membrane proteomes of self-renewing and differentiating human embryonic stem cells

    DEFF Research Database (Denmark)

    Prokhorova, Tatyana A; Rigbolt, Kristoffer T G; Johansen, Pia T;

    2009-01-01

    Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful quantitative proteomics platform for comprehensive characterization of complex biological systems. However, the potential of SILAC-based approaches has not been fully utilized in human embryonic stem cell (hESC) research...... embryonic stem cell lines. Of the 811 identified membrane proteins, six displayed significantly higher expression levels in the undifferentiated state compared with differentiating cells. This group includes the established marker CD133/Prominin-1 as well as novel candidates for hESC surface markers......: Glypican-4, Neuroligin-4, ErbB2, receptor-type tyrosine-protein phosphatase zeta (PTPRZ), and Glycoprotein M6B. Our study also revealed 17 potential markers of hESC differentiation as their corresponding protein expression levels displayed a dramatic increase in differentiated embryonic stem cell...

  20. Histochemical evidence for the differential surface labeling, uptake, and intracellular transport of a colloidal gold-labeled insulin complex by normal human blood cells

    International Nuclear Information System (INIS)

    A colloidal gold-labeled insulin-bovine serum albumin (GIA) reagent has been developed for the ultrastructural visualization of insulin binding sites on the cell surface and for tracing the pathway of intracellular insulin translocation. When applied to normal human blood cells, it was demonstrated by both visual inspection and quantitative analysis that the extent of surface labeling, as well as the rate and degree of internalization of the insulin complex, was directly related to cell type. Further, the pathway of insulin (GIA) transport via round vesicles and by tubulo-vesicles and saccules and its subsequent fate in the hemic cells was also related to cell variety. Monocytes followed by neutrophils bound the greatest amount of labeled insulin. The majority of lymphocytes bound and internalized little GIA, however, between 5-10% of the lymphocytes were found to bind considerable quantities of GIA. Erythrocytes rarely bound the labeled insulin complex, while platelets were noted to sequester large quantities of the GIA within their extracellular canalicular system. GIA uptake by the various types of leukocytic cells appeared to occur primarily by micropinocytosis and by the direct opening of cytoplasmic tubulo-vesicles and saccules onto the cell surface in regions directly underlying surface-bound GIA. Control procedures, viz., competitive inhibition of GIA labeling using an excess of unlabeled insulin in the incubation medium, preincubation of the GIA reagent with an antibody directed toward porcine insulin, and the incorporation of 125I-insulin into the GIA reagent, indicated the specificity and selectivity of the GIA histochemical procedure for the localization of insulin binding sites

  1. Epithelial Label-Retaining Cells Are Absent during Tooth Cycling in Salmo salar and Polypterus senegalus.

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    Sam Vandenplas

    Full Text Available The Atlantic salmon (Salmo salar and African bichir (Polypterus senegalus are both actinopterygian fish species that continuously replace their teeth without the involvement of a successional dental lamina. Instead, they share the presence of a middle dental epithelium: an epithelial tier enclosed by inner and outer dental epithelium. It has been hypothesized that this tier could functionally substitute for a successional dental lamina and might be a potential niche to house epithelial stem cells involved in tooth cycling. Therefore, in this study we performed a BrdU pulse chase experiment on both species to (1 determine the localization and extent of proliferating cells in the dental epithelial layers, (2 describe cell dynamics and (3 investigate if label-retaining cells are present, suggestive for the putative presence of stem cells. Cells proliferate in the middle dental epithelium, outer dental epithelium and cervical loop at the lingual side of the dental organ to form a new tooth germ. Using long chase times, both in S. salar (eight weeks and P. senegalus (eight weeks and twelve weeks, we could not reveal the presence of label-retaining cells in the dental organ. Immunostaining of P. senegalus dental organs for the transcription factor Sox2, often used as a stem cell marker, labelled cells in the zone of outer dental epithelium which grades into the oral epithelium (ODE transition zone and the inner dental epithelium of a successor only. The location of Sox2 distribution does not provide evidence for epithelial stem cells in the dental organ and, more specifically, in the middle dental epithelium. Comparison of S. salar and P. senegalus reveals shared traits in tooth cycling and thus advances our understanding of the developmental mechanism that ensures lifelong replacement.

  2. Epithelial Label-Retaining Cells Are Absent during Tooth Cycling in Salmo salar and Polypterus senegalus.

    Science.gov (United States)

    Vandenplas, Sam; Willems, Maxime; Witten, P Eckhard; Hansen, Tom; Fjelldal, Per Gunnar; Huysseune, Ann

    2016-01-01

    The Atlantic salmon (Salmo salar) and African bichir (Polypterus senegalus) are both actinopterygian fish species that continuously replace their teeth without the involvement of a successional dental lamina. Instead, they share the presence of a middle dental epithelium: an epithelial tier enclosed by inner and outer dental epithelium. It has been hypothesized that this tier could functionally substitute for a successional dental lamina and might be a potential niche to house epithelial stem cells involved in tooth cycling. Therefore, in this study we performed a BrdU pulse chase experiment on both species to (1) determine the localization and extent of proliferating cells in the dental epithelial layers, (2) describe cell dynamics and (3) investigate if label-retaining cells are present, suggestive for the putative presence of stem cells. Cells proliferate in the middle dental epithelium, outer dental epithelium and cervical loop at the lingual side of the dental organ to form a new tooth germ. Using long chase times, both in S. salar (eight weeks) and P. senegalus (eight weeks and twelve weeks), we could not reveal the presence of label-retaining cells in the dental organ. Immunostaining of P. senegalus dental organs for the transcription factor Sox2, often used as a stem cell marker, labelled cells in the zone of outer dental epithelium which grades into the oral epithelium (ODE transition zone) and the inner dental epithelium of a successor only. The location of Sox2 distribution does not provide evidence for epithelial stem cells in the dental organ and, more specifically, in the middle dental epithelium. Comparison of S. salar and P. senegalus reveals shared traits in tooth cycling and thus advances our understanding of the developmental mechanism that ensures lifelong replacement. PMID:27049953

  3. Magnetic separation of encapsulated islet cells labeled with superparamagnetic iron oxide nano particles.

    Science.gov (United States)

    Mettler, Esther; Trenkler, Anja; Feilen, Peter J; Wiegand, Frederik; Fottner, Christian; Ehrhart, Friederike; Zimmermann, Heiko; Hwang, Yong Hwa; Lee, Dong Yun; Fischer, Stefan; Schreiber, Laura M; Weber, Matthias M

    2013-01-01

    Islet cell transplantation is a promising option for the restoration of normal glucose homeostasis in patients with type 1 diabetes. Because graft volume is a crucial issue in islet transplantations for patients with diabetes, we evaluated a new method for increasing functional tissue yield in xenogeneic grafts of encapsulated islets. Islets were labeled with three different superparamagnetic iron oxide nano particles (SPIONs; dextran-coated SPION, siloxane-coated SPION, and heparin-coated SPION). Magnetic separation was performed to separate encapsulated islets from the empty capsules, and cell viability and function were tested. Islets labeled with 1000 μg Fe/ml dextran-coated SPIONs experienced a 69.9% reduction in graft volume, with a 33.2% loss of islet-containing capsules. Islets labeled with 100 μg Fe/ml heparin-coated SPIONs showed a 46.4% reduction in graft volume, with a 4.5% loss of capsules containing islets. No purification could be achieved using siloxane-coated SPIONs due to its toxicity to the primary islets. SPION labeling of islets is useful for transplant purification during islet separation as well as in vivo imaging after transplantation. Furthermore, purification of encapsulated islets can also reduce the volume of the encapsulated islets without impairing their function by removing empty capsules.

  4. Label-free separation of human embryonic stem cells (hESCs) and their cardiac derivatives using Raman spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Chan, J W; Lieu, D K; Huser, T R; Li, R A

    2008-09-08

    Self-renewable, pluripotent human embryonic stem cells (hESCs) can be differentiated into cardiomyocytes (CMs), providing an unlimited source of cells for transplantation therapies. However, unlike certain cell lineages such as hematopoietic cells, CMs lack specific surface markers for convenient identification, physical separation, and enrichment. Identification by immunostaining of cardiac-specific proteins such as troponin requires permeabilization, which renders the cells unviable and non-recoverable. Ectopic expression of a reporter protein under the transcriptional control of a heart-specific promoter for identifying hESC-derived CMs (hESC-CMs) is useful for research but complicates potential clinical applications. The practical detection and removal of undifferentiated hESCs in a graft, which may lead to tumors, is also critical. Here, we demonstrate a non-destructive, label-free optical method based on Raman scattering to interrogate the intrinsic biochemical signatures of individual hESCs and their cardiac derivatives, allowing cells to be identified and classified. By combining the Raman spectroscopic data with multivariate statistical analysis, our results indicate that hESCs, human fetal left ventricular CMs, and hESC-CMs can be identified by their intrinsic biochemical characteristics with an accuracy of 96%, 98% and 66%, respectively. The present study lays the groundwork for developing a systematic and automated method for the non-invasive and label-free sorting of (i) high-quality hESCs for expansion, and (ii) ex vivo CMs (derived from embryonic or adult stem cells) for cell-based heart therapies.

  5. Label-free detection of liver cancer cells by aptamer-based microcantilever biosensor.

    Science.gov (United States)

    Chen, Xuejuan; Pan, Yangang; Liu, Huiqing; Bai, Xiaojing; Wang, Nan; Zhang, Bailin

    2016-05-15

    Liver cancer is one of the most common and highly malignant cancers in the world. There are no effective therapeutic options if an early liver cancer diagnosis is not achieved. In this work, detection of HepG2 cells by label-free microcantilever array aptasensor was developed. The sensing microcantilevers were functionalized by HepG2 cells-specific aptamers. Meanwhile, to eliminate the interferences induced by the environment, the reference microcantilevers were modified with 6-mercapto-1-hexanol self-assembled monolayers. The aptasensor exhibits high specificity over not only human liver normal cells, but also other cancer cells of breast, bladder, and cervix tumors. The linear relation ranges from 1×10(3) to 1×10(5)cells/mL, with a detection limit of 300 cells/mL (S/N=3). Our work provides a simple method for detection of liver cancer cells with advantages in terms of simplicity and stability.

  6. Label-free isolation and deposition of single bacterial cells from heterogeneous samples for clonal culturing.

    Science.gov (United States)

    Riba, J; Gleichmann, T; Zimmermann, S; Zengerle, R; Koltay, P

    2016-01-01

    The isolation and analysis of single prokaryotic cells down to 1 μm and less in size poses a special challenge and requires micro-engineered devices to handle volumes in the picoliter to nanoliter range. Here, an advanced Single-Cell Printer (SCP) was applied for automated and label-free isolation and deposition of bacterial cells encapsulated in 35 pl droplets by inkjet-like printing. To achieve this, dispenser chips to generate micro droplets have been fabricated with nozzles 20 μm in size. Further, the magnification of the optical system used for cell detection was increased. Redesign of the optical path allows for collision-free addressing of any flat substrate since no compartment protrudes below the nozzle of the dispenser chip anymore. The improved system allows for deterministic isolation of individual bacterial cells. A single-cell printing efficiency of 93% was obtained as shown by printing fluorescent labeled E. coli. A 96-well plate filled with growth medium is inoculated with single bacteria cells on average within about 8 min. Finally, individual bacterial cells from a heterogeneous sample of E. coli and E. faecalis were isolated for clonal culturing directly on agar plates in user-defined array geometry. PMID:27596612

  7. Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

    International Nuclear Information System (INIS)

    We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.

  8. Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei, E-mail: biehzw@nus.edu.sg [Optical Bioimaging Laboratory, Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, Singapore 117576 (Singapore)

    2014-09-08

    We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.

  9. Near-Infrared Imaging of Adoptive Immune Cell Therapy in Breast Cancer Model Using Cell Membrane Labeling

    OpenAIRE

    Youniss, Fatma M.; Gobalakrishnan Sundaresan; Graham, Laura J.; Li Wang; Berry, Collin R.; Dewkar, Gajanan K.; Purnima Jose; Bear, Harry D; Jamal Zweit

    2014-01-01

    The overall objective of this study is to non-invasively image and assess tumor targeting and retention of directly labeled T-lymphocytes following their adoptive transfer in mice. T-lymphocytes obtained from draining lymph nodes of 4T1 (murine breast cancer cell) sensitized BALB/C mice were activated in-vitro with Bryostatin/Ionomycin for 18 hours, and were grown in the presence of Interleukin-2 for 6 days. T-lymphocytes were then directly labeled with 1,1-dioctadecyltetramethyl indotricarbo...

  10. In vivo MR tracking of magnetically labeled mesenchymal stem cells in rat liver after intrasplenic transplantation

    International Nuclear Information System (INIS)

    Objective: To evaluate the 1.5-T magnetic resonance imaging system to depict and track in vivo of magnetically labeled bone mesenchymal stem cells (BMSCs) in rat liver after intrasplenic transplantation. Methods: Rat BMSCs were isolated, purified, expanded and then incubated with home synthesized Fe2O3-PLL. Prussian blue stain was performed for showing intracellular irons. Acute liver damage was induced by subcutaneous injection of carbon tetrachloride in 12 recipient rats. The cells allotransplantation was performed by intrasplenic injection with magnetically labeled (experimental group, n=6) or unlabeled BMSCs (control group, n=6). Serial MRI were performed before and 3 hours, 3, 7, 14 days after transplantation. Signal-to-noise ratio (SNR) of liver on T2*-weighted MR imaging obtained before and after injection were measured and compared. MR imaging findings were compared histologically with histology. Results: Rats BMSCs could be effectively labeled and the labeling efficiency was almost 100%. Iron-containing intracytoplasmatic vesicles could be observed clearly with prussian blue staining. SNR of rat livers in experimental group and control group before and 3 hours, 3, 7, 14 days after transplantation were 19.53±2.30, 3.28±1.06, 7.34±2.10, 10.25±3.96, 15.50±3.73; 20.20±4.35, 21.20±4.43, 19.13±2.80, 21.43±5.45, 19.07±4.80, respectively. SNR decreased significantly in the experimental group liver 3 hours, 3, 7 days after injection of BMSCs (Dunnett test, P0.05). In control group, SNR demonstrated no significant differences among different time points (ANOVA, P>0.05). Results of histological analysis confirmed homing of labeled BMSCs in liver, primarily distributing in areas around centrolobular vein. Conclusion: The BMSCs can be effectively labeled with Fe2O3-PLL. 1.5-T MR imaging can monitor in vivo of magnetically labeled BMSCs in liver after intrasplenic transplantation. It potentially opens a new area of investigation for delivering stem cells

  11. Comparison between 125IUdR and 51Cr as cell labels in investigations of tumor cell migration

    DEFF Research Database (Denmark)

    Basse, P; Hokland, P; Hokland, M

    1991-01-01

    YAC-1 tumor cells double-labeled with Na2[51Cr]O4 [51Cr] and [125I]iododeoxyuridine [125IUdR] were injected intravenously into Balb/c mice in order to investigate their migration and fate 0-4 h after the injection. Whereas the clearance of tumor cells from the lung tissue was similar as judged...... with both labels, the kinetics of isotope uptake in the liver were strikingly different. Thus, retention of 51Cr in the liver was very high compared to a much lower and only transient retention of 125I. A higher retention of non-tumor cell-associated 51Cr was also observed in most other organs, resulting...... in overestimation of the number of viable tumor cells in these organs. Moreover, a marked spontaneous release (greater than 10% after 12 h) makes 51Cr less suitable as a cell label than 125IUdR. On the other hand, we found that the release of 125I from dead cells in vivo depends at least partially on host factors...

  12. Autoradiographic study on the incorporation of carbon-14 labeled formate and adenine into nucleic acid in blood-forming cells

    International Nuclear Information System (INIS)

    The incorporation of [14C]formate and [8-14C]adenine into nucleic acid in blood-forming cells was studied by the autoradiographic technique. The isotopic markers were injected subcutaneously into young rats weighting from 100 to 150 g three times every 24 hours and the animals were examined 3 hours after the last injection. In the case of [14C]formate injection, erythroblasts exhibited extremely strong labeling in contrast to weaker labeling of other blood-forming cells. In the case of [14C]adenine administration, on the other hand, immature cells of the granuclocytic series as well as immature reticulum cells (proliferating cells of reticular tissue) were much more heavily labeled than were other blood-forming cells, particularly the erythroblasts which revealed weak or no labeling. By digestion or extraction of DNA, RNA or both from cells with DNase, RNase or hot 10% perchloric acid treatment, respectively, it was confirmed that the observed heavy labeling of any type of cells with either [14C]formate or [14C]adenine was due chiefly to incorporation of the radioactive materials into nuclear DNA. The present results are discussed together with the findings of earlier studies on lymphoid cells which indicate that, in certain cell types, the patterns of [3H]deoxycytidine labeling differ considerably from the corresponding patterns of [3H]deoxycytidine labeling. The present and earlier findings provide evidence to substantiate that, among blood-forming cells, there are considerable variations in the labeling patterns of nuclear DNA depending on differences in the radioactive DNA precursors used as well as in the cell types. (author)

  13. Label-free characterization of white blood cells by measuring 3D refractive index maps

    CERN Document Server

    Yoon, Jonghee; Park, HyunJoo; Choi, Chulhee; Jang, Seongsoo; Park, YongKeun

    2015-01-01

    The characterization of white blood cells (WBCs) is crucial for blood analyses and disease diagnoses. However, current standard techniques rely on cell labeling, a process which imposes significant limitations. Here we present three-dimensional (3D) optical measurements and the label-free characterization of mouse WBCs using optical diffraction tomography. 3D refractive index (RI) tomograms of individual WBCs are constructed from multiple two-dimensional quantitative phase images of samples illuminated at various angles of incidence. Measurements of the 3D RI tomogram of WBCs enable the separation of heterogeneous populations of WBCs using quantitative morphological and biochemical information. Time-lapse tomographic measurements also provide the 3D trajectory of micrometer-sized beads ingested by WBCs. These results demonstrate that optical diffraction tomography can be a useful and versatile tool for the study of WBCs.

  14. Mathematical analysis of /sup 51/Cr-labelled red cell survival curves in congenital haemolytic anaemias

    Energy Technology Data Exchange (ETDEWEB)

    Kasfiki, A.G.; Antipas, S.E.; Dimitriou, P.A.; Gritzali, F.A.; Melissinos, K.G.

    1982-04-01

    The parameters of /sup 51/Cr labelled red cell survival curves were calculated in 26 patients with homozygous ..beta..-thalassaemia, 8 with sickle-cell anaemia and 3 with s-..beta..-thalassaemia, using a non-linear weighted least squares analysis computer program. In thalassaemic children the calculated parameters denote that the shorting of the mean cell life is due to early senescence alone, while there is some evidence that in thalassaemic adults additional extracellular destruction mechanisms participate as well. Red cell survival curves from patients with sickle-cell anaemia and s-..beta..-thalassaemia resemble each other, while their parameters indicate an initial rapid loss of radioactivity, early senescence and the presence of extracellular red cell destruction factors.

  15. Mathematical analysis of 51Cr-labelled red cell survival curves in congenital haemolytic anaemias

    International Nuclear Information System (INIS)

    The parameters of 51Cr labelled red cell survival curves were calculated in 26 patients with homozygous β-thalassaemia, 8 with sickle-cell anaemia and 3 with s-β-thalassaemia, using a non-linear weighted least squares analysis computer program. In thalassaemic children the calculated parameters denote that the shorting of the mean cell life is due to early senescence alone, while there is some evidence that in thalassaemic adults additional extracellular destruction mechanisms participate as well. Red cell survival curves from patients with sickle-cell anaemia and s-β-thalassaemia resemble each other, while their parameters indicate an initial rapid loss of radioactivity, early senescence and the presence of extracellular red cell destruction factors. (orig.)

  16. A Simple Method for Labeling Human Embryonic Stem Cells Destined to Lose Undifferentiated Potency.

    Science.gov (United States)

    Kumagai, Ayako; Suga, Mika; Yanagihara, Kana; Itoh, Yumi; Takemori, Hiroshi; Furue, Miho K

    2016-03-01

    Mitochondrial oxidative phosphorylation is a major source of cellular ATP. Its usage as an energy source varies, not only according to the extracellular environment, but also during development and differentiation, as indicated by the reported changes in the flux ratio of glycolysis to oxidative phosphorylation during embryonic stem (ES) cell differentiation. The fluorescent probe JC-1 allows visualization of changes in the mitochondrial membrane potential produced by oxidative phosphorylation. Strong JC-1 signals were localized in the differentiated cells located at the edge of H9 ES colonies that expressed vimentin, an early differentiation maker. The JC-1 signals were further intensified when individual adjacent colonies were in contact with each other. Time-lapse analyses revealed that JC-1-labeled H9 cells under an overconfluent condition were highly differentiated after subculture, suggesting that monitoring oxidative phosphorylation in live cells might facilitate the prediction of induced pluripotent stem cells, as well as ES cells, that are destined to lose their undifferentiated potency.

  17. Rare-Cell Enrichment by a Rapid, Label-Free, Ultrasonic Isopycnic Technique for Medical Diagnostics**

    Science.gov (United States)

    Bourquin, Yannyk; Syed, Abeer; Reboud, Julien; Ranford-Cartwright, Lisa C; Barrett, Michael P; Cooper, Jonathan M

    2014-01-01

    One significant challenge in medical diagnostics lies in the development of label-free methods to separate different cells within complex biological samples. Here we demonstrate a generic, low-power ultrasonic separation technique, able to enrich different cell types based upon their physical properties. For malaria, we differentiate between infected and non-infected red blood cells in a fingerprick-sized drop of blood. We are able to achieve an enrichment of circulating cells infected by the ring stage of the parasite over nonparasitized red blood cells by between two and three orders of magnitude in less than 3 seconds (enabling detection at parasitemia levels as low as 0.0005 %). In a second example, we also show that our methods can be used to enrich different cell types, concentrating Trypanosoma in blood at very low levels of infection, on disposable, low-cost chips. PMID:24677583

  18. Rare-cell enrichment by a rapid, label-free, ultrasonic isopycnic technique for medical diagnostics.

    Science.gov (United States)

    Bourquin, Yannyk; Syed, Abeer; Reboud, Julien; Ranford-Cartwright, Lisa C; Barrett, Michael P; Cooper, Jonathan M

    2014-05-26

    One significant challenge in medical diagnostics lies in the development of label-free methods to separate different cells within complex biological samples. Here we demonstrate a generic, low-power ultrasonic separation technique, able to enrich different cell types based upon their physical properties. For malaria, we differentiate between infected and non-infected red blood cells in a fingerprick-sized drop of blood. We are able to achieve an enrichment of circulating cells infected by the ring stage of the parasite over nonparasitized red blood cells by between two and three orders of magnitude in less than 3 seconds (enabling detection at parasitemia levels as low as 0.0005%). In a second example, we also show that our methods can be used to enrich different cell types, concentrating Trypanosoma in blood at very low levels of infection, on disposable, low-cost chips. PMID:24677583

  19. Cell proliferation in the Drosophila adult brain revealed by clonal analysis and bromodeoxyuridine labelling

    OpenAIRE

    Brand Andrea H; Egger Boris; von Trotha Jakob W

    2009-01-01

    Abstract Background The production of new neurons during adulthood and their subsequent integration into a mature central nervous system have been shown to occur in all vertebrate species examined to date. However, the situation in insects is less clear and, in particular, it has been reported that there is no proliferation in the Drosophila adult brain. Results We report here, using clonal analysis and 5'-bromo-2'-deoxyuridine (BrdU) labelling, that cell proliferation does occur in the Droso...

  20. Tritiated thymidine-labeled bronchioloalveolar cells and radiation dose following inhalation of plutonium in rats

    International Nuclear Information System (INIS)

    The goal of this study is to show the relationship of inhaled Pu particle distribution and alveolar-bronchiolar target-cell response with respect to the formation of pulmonary carcinoma. The proliferation of type 2 alveolar epithelium and nonciliated bronchiolar epithelium appears critical in the induction of lung tumors associated from inhaled 239PuO2. Female, Wistar rats were either sham-exposed (40 rats) or given a single inhalation to 169Yb-239PuO2 (99 rats, ILB, 3.9 +/- 1.2 kBq) and examined at 20 time intervals from 1 day to 700 days postexposure for Pu particle distribution in airways by SEM quantitative autoradiography and for cell labeling with tritiated thymidine. Initially, deposited Pu particles were rapidly cleared from the surface of the trachea, bronchi, and bronchioles within a few days. Thereafter, about 5 times more alpha track exposure to the bronchiolar epithelium was delivered from Pu particles found in peribronchiolar alveoli than from Pu particles being cleared from bronchiolar surfaces. Exposure of bronchiolar epithelium at later times was due mostly to the formation of peribronchiolar Pu particle aggregates. A maximal increase in labeled alveolar wall cells was seen at 60 days after exposure, decreasing gradually to control levels by 400 days. Cell labeling in focal alveolar regions of Pu aggregation was about 5 fold higher. Increased bronchiolar epithelium labeling appeared in two phases. The first phase was seen 15 days after exposure, associated with initial deposition and clearance of Pu particles. The second phase slowly reached a maximum at 250 days and was associated with peribronchiolar Pu aggregate formation. The temporal-spatial dose-distribution pattern for inhaled Pu particles is an important aspect of Pu-induced pulmonary carcinogenesis

  1. An economic approach to efficient isotope labeling in insect cells using homemade 15N-, 13C- and 2H-labeled yeast extracts

    International Nuclear Information System (INIS)

    Heterologous expression of proteins in insect cells is frequently used for crystallographic structural studies due to the high yields even for challenging proteins requiring the eukaryotic protein processing capabilities of the host. However for NMR studies, the need for isotope labeling poses extreme challenges in eukaryotic hosts. Here, we describe a robust method to achieve uniform protein 15N and 13C labeling of up to 90 % in baculovirus-infected insect cells. The approach is based on the production of labeled yeast extract, which is subsequently supplemented to insect cell growth media. The method also allows deuteration at levels of >60 % without decrease in expression yield. The economic implementation of the labeling procedures into a standard structural biology laboratory environment is described in a step-by-step protocol. Applications are demonstrated for a variety of NMR experiments using the Abelson kinase domain, GFP, and the beta-1 adrenergic receptor as examples. Deuterated expression of the latter provides spectra of very high quality of a eukaryotic G-protein coupled receptor

  2. An economic approach to efficient isotope labeling in insect cells using homemade {sup 15}N-, {sup 13}C- and {sup 2}H-labeled yeast extracts

    Energy Technology Data Exchange (ETDEWEB)

    Opitz, Christian; Isogai, Shin; Grzesiek, Stephan, E-mail: Stephan.Grzesiek@unibas.ch [University of Basel, Focal Area Structural Biology and Biophysics, Biozentrum (Switzerland)

    2015-07-15

    Heterologous expression of proteins in insect cells is frequently used for crystallographic structural studies due to the high yields even for challenging proteins requiring the eukaryotic protein processing capabilities of the host. However for NMR studies, the need for isotope labeling poses extreme challenges in eukaryotic hosts. Here, we describe a robust method to achieve uniform protein {sup 15}N and {sup 13}C labeling of up to 90 % in baculovirus-infected insect cells. The approach is based on the production of labeled yeast extract, which is subsequently supplemented to insect cell growth media. The method also allows deuteration at levels of >60 % without decrease in expression yield. The economic implementation of the labeling procedures into a standard structural biology laboratory environment is described in a step-by-step protocol. Applications are demonstrated for a variety of NMR experiments using the Abelson kinase domain, GFP, and the beta-1 adrenergic receptor as examples. Deuterated expression of the latter provides spectra of very high quality of a eukaryotic G-protein coupled receptor.

  3. An economic approach to efficient isotope labeling in insect cells using homemade 15N-, 13C- and 2H-labeled yeast extracts.

    Science.gov (United States)

    Opitz, Christian; Isogai, Shin; Grzesiek, Stephan

    2015-07-01

    Heterologous expression of proteins in insect cells is frequently used for crystallographic structural studies due to the high yields even for challenging proteins requiring the eukaryotic protein processing capabilities of the host. However for NMR studies, the need for isotope labeling poses extreme challenges in eukaryotic hosts. Here, we describe a robust method to achieve uniform protein (15)N and (13)C labeling of up to 90 % in baculovirus-infected insect cells. The approach is based on the production of labeled yeast extract, which is subsequently supplemented to insect cell growth media. The method also allows deuteration at levels of >60 % without decrease in expression yield. The economic implementation of the labeling procedures into a standard structural biology laboratory environment is described in a step-by-step protocol. Applications are demonstrated for a variety of NMR experiments using the Abelson kinase domain, GFP, and the beta-1 adrenergic receptor as examples. Deuterated expression of the latter provides spectra of very high quality of a eukaryotic G-protein coupled receptor. PMID:26070442

  4. Toward label-free Raman-activated cell sorting of cardiomyocytes derived from human embryonic stem cells

    Science.gov (United States)

    Pascut, Flavius C.; Goh, Huey T.; George, Vinoj; Denning, Chris; Notingher, Ioan

    2011-04-01

    Raman micro-spectroscopy (RMS) has been recently proposed for label-free phenotypic identification of human embryonic stem cells (hESC)-derived cardiomyocytes. However, the methods used for measuring the Raman spectra led to acquisition times of minutes per cell, which is prohibitive for rapid cell sorting applications. In this study we evaluated two measurement strategies that could reduce the measurement time by a factor of more than 100. We show that sampling individual cells with a laser beam focused to a line could eliminate the need of cell raster scanning and achieve high prediction accuracies (>95% specificity and >96% sensitivity) with acquisition times ~5 seconds per cell. However, the use of commercially-available higher power lasers could potentially lead to sorting speeds of ~10 cells per s. This would start to progress RMS to the field of cell sorting for applications such as enrichment and purification of hESC-derived cardiomyocytes.

  5. Fluorescent labelling of DNA on superparamagnetic nanoparticles by a perylene bisimide derivative for cell imaging

    Energy Technology Data Exchange (ETDEWEB)

    Maltas, Esra, E-mail: maltasesra@gmail.com [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey); Malkondu, Sait [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey); Uyar, Pembegul [Selcuk University, Faculty of Science, Department of Biology, 42075 Konya (Turkey); Selcuk University, Advanced Technology Research and Application Center, Konya (Turkey); Ozmen, Mustafa [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey)

    2015-03-01

    N,N′-Bis[tris-(2-aminoethyl) amine]-3,4,9,10-perylenetetracarboxylic diimide (PBI-TRIS), nonfluorescent dye was used to fluorescent labelling of DNA. For this aim, (3-aminopropyl) triethoxysilane (APTS) modified superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized to provide a suitable surface for binding of DNA. Amine functionalized nanoparticles showed a high immobilization capacity (82.70%) at 25 mg of nanoparticle concentration for Calf thymus DNA. Binding capacity of PBI-TRIS to DNA-SPION was also found as 1.93 μM on 25 mg of nanoparticles by using UV–vis spectroscopy. Binding of PBI-TRIS to DNA onto nanoparticles was also characterized by scanning electron microscopy and infrared spectroscopy. The confocal images of PBI-TRIS labelled DNA-SPION and breast cells were taken at 488 and 561.7 nm of excitation wavelengths. Cell image was also compared with a commercial dye, DAPI at 403.7 nm of excitation wavelength. Results showed that PBI-TRIS can be used for cell staining. - Highlights: • Functionalized SPIONs were synthesized and treated with DNA. • The binding of PBI-TRIS with DNA was studied. • The image of PBI-TRIS labelled DNA-SPION was detected by a confocal microscope.

  6. Fluorescent labelling of DNA on superparamagnetic nanoparticles by a perylene bisimide derivative for cell imaging

    International Nuclear Information System (INIS)

    N,N′-Bis[tris-(2-aminoethyl) amine]-3,4,9,10-perylenetetracarboxylic diimide (PBI-TRIS), nonfluorescent dye was used to fluorescent labelling of DNA. For this aim, (3-aminopropyl) triethoxysilane (APTS) modified superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized to provide a suitable surface for binding of DNA. Amine functionalized nanoparticles showed a high immobilization capacity (82.70%) at 25 mg of nanoparticle concentration for Calf thymus DNA. Binding capacity of PBI-TRIS to DNA-SPION was also found as 1.93 μM on 25 mg of nanoparticles by using UV–vis spectroscopy. Binding of PBI-TRIS to DNA onto nanoparticles was also characterized by scanning electron microscopy and infrared spectroscopy. The confocal images of PBI-TRIS labelled DNA-SPION and breast cells were taken at 488 and 561.7 nm of excitation wavelengths. Cell image was also compared with a commercial dye, DAPI at 403.7 nm of excitation wavelength. Results showed that PBI-TRIS can be used for cell staining. - Highlights: • Functionalized SPIONs were synthesized and treated with DNA. • The binding of PBI-TRIS with DNA was studied. • The image of PBI-TRIS labelled DNA-SPION was detected by a confocal microscope

  7. Diagnosis of infection by preoperative scintigraphy with indium-labeled white blood cells

    International Nuclear Information System (INIS)

    Scintigraphy with indium-labeled white blood cells has been reported to be sensitive and specific in the diagnosis of low-grade sepsis of the musculoskeletal system. We reviewed the records of fifty patients who had suspected osteomyelitis or suspected infection about a total joint prosthesis and who underwent scintigraphy with technetium-99m methylene diphosphonate and scintigraphy with indium-111 oxine-labeled white blood cells before an open surgical procedure. Any patient who received preoperative antibiotics was not included in the study. For all of the patients, gram-stain examination of smears, evaluation of a culture of material from the operative site, and histological examination were done. The patients were divided into two groups. Group I was composed of twenty-four patients, each of whom had a prosthesis in place and complained of pain. Group II was composed of twenty-six patients for whom a diagnosis of chronic osteomyelitis had to be considered. With the indium scans alone, there was only one false-negative result (in Group II), but there were eighteen false-positive results (eight patients in Group II and ten patients in Group I). Although scintigraphy with indium-labeled white blood cells is quite sensitive, it is not specific in detecting chronic osteomyelitis; a negative scan should be considered highly suggestive that osteomyelitis is not present. Specificity can be increased by interpreting the indium scan in conjunction with the technetium scan

  8. The effect of an external magnetic force on cell adhesion and proliferation of magnetically labeled mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Nakamae Toshio

    2010-02-01

    Full Text Available Abstract Background As the strategy for tissue regeneration using mesenchymal stem cells (MSCs for transplantation, it is necessary that MSCs be accumulated and kept in the target area. To accumulate MSCs effectively, we developed a novel technique for a magnetic targeting system with magnetically labeled MSCs and an external magnetic force. In this study, we examined the effect of an external magnetic force on magnetically labeled MSCs in terms of cell adhesion and proliferation. Methods Magnetically labeled MSCs were plated at the bottom of an insert under the influence of an external magnetic force for 1 hour. Then the inserts were turned upside down for between 1 and 24 hours, and the number of MSCs which had fallen from the membrane was counted. The gene expression of MSCs affected magnetic force was analyzed with microarray. In the control group, the same procedure was done without the external magnetic force. Results At 1 hour after the inserts were turned upside down, the average number of fallen MSCs in the magnetic group was significantly smaller than that in the control group, indicating enhanced cell adhesion. At 24 hours, the average number of fallen MSCs in the magnetic group was also significantly smaller than that in control group. In the magnetic group, integrin alpha2, alpha6, beta3 BP, intercellular adhesion molecule-2 (ICAM-2, platelet/endothelial cell adhesion molecule-1 (PECAM-1 were upregulated. At 1, 2 and 3 weeks after incubation, there was no statistical significant difference in the numbers of MSCs in the magnetic group and control group. Conclusions The results indicate that an external magnetic force for 1 hour enhances cell adhesion of MSCs. Moreover, there is no difference in cell proliferation after using an external magnetic force on magnetically labeled MSCs.

  9. Magnetic Labelling of Mesenchymal Stem Cells with Iron-Doped Hydroxyapatite Nanoparticles as Tool for Cell Therapy.

    Science.gov (United States)

    Panseri, Silvia; Montesi, Monica; Iafisco, Michele; Adamiano, Alessio; Ghetti, Martina; Cenacchi, Giovanna; Tampieri, Anna

    2016-05-01

    Superparamagnetic nanoparticles offer several opportunities in nanomedicine and magnetic cell targeting. They are considered to be an extremely promising approach for the translation of cell-based therapies from the laboratory to clinical studies. In fact, after injection, the magnetic labeled cells could be driven by a static magnetic field and localized to the target site where they can perform their specific role. In this study, innovative iron-doped hydroxyapatite nanoparticles (FeHA NPs) were tested with mesenchymal stem cells (MSCs) as tools for cell therapy. Results showed that FeHA NPs could represent higher cell viability in'respect to commercial superparamagnetic iron oxide nanoparticles (SPION) at four different concentrations ranging from 10 μg/ml up to 200 μg/ml and would also upregulate an early marker involved in commitment and differentiation of MSCs. Moreover, FeHA NPs were uptaken without negatively affecting the cell behavior and their ultrastructure. Thus obtained magnetic cells were easily guided by application of a static magnetic field. This work demonstrates the promising opportunities of FeHA NPs in MSCs labeling due to the unique features of fast degradation and very low iron content of FeHA NPs compared to SPIONs. Likewise, due to the intrinsic properties of FeHA NPs, this approach could be simply transferred to different cell types as an effective magnetic carrier of drugs, growth factors, miRNA, etc., offering favorable prospects in nanomedicine.

  10. 2D light scattering static cytometry for label-free single cell analysis with submicron resolution.

    Science.gov (United States)

    Xie, Linyan; Yang, Yan; Sun, Xuming; Qiao, Xu; Liu, Qiao; Song, Kun; Kong, Beihua; Su, Xuantao

    2015-11-01

    Conventional optical cytometric techniques usually measure fluorescence or scattering signals at fixed angles from flowing cells in a liquid stream. Here we develop a novel cytometer that employs a scanning optical fiber to illuminate single static cells on a glass slide, which requires neither microfluidic fabrication nor flow control. This static cytometric technique measures two dimensional (2D) light scattering patterns via a small numerical aperture (0.25) microscope objective for label-free single cell analysis. Good agreement is obtained between the yeast cell experimental and Mie theory simulated patterns. It is demonstrated that the static cytometer with a microscope objective of a low resolution around 1.30 μm has the potential to perform high resolution analysis on yeast cells with distributed sizes. The capability of the static cytometer for size determination with submicron resolution is validated via measurements on standard microspheres with mean diameters of 3.87 and 4.19 μm. Our 2D light scattering static cytometric technique may provide an easy-to-use, label-free, and flow-free method for single cell diagnostics.

  11. Label-free continuous cell sorter with specifically adhesive oblique micro-grooves

    International Nuclear Information System (INIS)

    We report the development of a label-free continuous cell sorting method based on specific adhesivity between cells and surface-immobilized adhesion molecules. The separation of cells is induced by cross-flow adhesive force on micron-sized stripes with adhesion molecules immobilized on the surface. In order to accurately form the adhesive stripes on a microchannel wall, 1 µm wide micro-grooves are fabricated at a certain angle with respect to the flow direction using direct electron-beam lithography. Amino-functionalized parylene is used as the groove surface material, and streptavidin is immobilized on the entire surface, resulting in a surface with periodic adhesive patterns. The effectiveness of the proposed cell sorting principle is verified by flow-through experiments using functionalized particles as model cells. Measurements of the motion of biotin-coated microparticles show that the particles decelerated by specific adhesivity are displaced in the cross-flow direction. The observed cross-flow displacement is around 0.8% of the streamwise travelling distance. It is also shown that the rate of cross-flow displacement is independent of the flow rate or the stripe angle. Finally, it is demonstrated that a mixture of streptavidin- and biotin-coated microparticles can be completely separated after flowing over a 20 mm long patterned surface. The proposed label-free continuous lateral separation scheme has a wide range of potential applications for separation of cells which could not be distinguished by size or separated using dielectric forces

  12. Connected components labeling for giga-cell multi-categorical rasters

    Science.gov (United States)

    Netzel, Pawel; Stepinski, Tomasz F.

    2013-09-01

    Labeling of connected components in an image or a raster of non-imagery data is a fundamental operation in fields of pattern recognition and machine intelligence. The bulk of effort devoted to designing efficient connected components labeling (CCL) algorithms concentrated on the domain of binary images where labeling is required for a computer to recognize objects. In contrast, in the Geographical Information Science (GIS) a CCL algorithm is mostly applied to multi-categorical rasters in order to either convert a raster to a shapefile, or for statistical characterization of individual clumps. Recently, it has become necessary to label connected components in very large, giga-cell size, multi-categorical rasters but performance of existing CCL algorithms lacks sufficient speed to accomplish such task. In this paper we present a modification to the popular two-scan CCL algorithm that enables labeling of giga-cell size, multi-categorical rasters. Our approach is to apply a divide-and-conquer technique coupled with parallel processing to a standard two-scan algorithm. For specificity, we have developed a variant of a standard CCL algorithm implemented as r.clump in GRASS GIS. We have established optimal values of data blocks (stemming from the divide-and-conquer technique) and optimal number of computational threads (stemming from parallel processing) for a new algorithm called r.clump3p. The performance of the new algorithm was tested on a series of rasters up to 160 Mcells in size; for largest size test raster a speed up over the original algorithm is 74 times. Finally, we have applied the new algorithm to the National Land Cover Dataset 2006 raster with 1.6×1010 cells. Labeling this raster took 39 h using two-processors, 16 cores computer and resulted in 221,718,501 clumps. Estimated speed up over the original algorithm is 450 times. The r.clump3p works within the GRASS environment and is available in the public domain.

  13. Labeling and imaging of human mesenchymal stem cells with quantum dot bioconjugates during proliferation and osteogenic differentiation in long term.

    Science.gov (United States)

    Shah, B; Clark, P; Stroscio, M; Mao, J

    2006-01-01

    Quantum dots (QDs) are semiconductor nanocrystals that serve as promising alternatives to organic dyes for cell labeling. Because of their unique spectral, physical and chemical properties, QDs are useful for concurrently monitoring several intercellular and intracellular interactions in live normal cells and cancer cells over periods ranging from less than a second to over several days (several divisions of cells). Here, peptide CGGGRGD is immobilized on CdSe-ZnS QDs coated with carboxyl groups by cross linking with amine groups. These conjugates are directed by the peptide to bind with selected integrins on the membrane of human Mesenchymal stem cells. Upon overnight incubation with optimal concentration, QDs effectively labeled all the cells. Here, we report long-term labeling of human bone-marrow-derived mesenchymal stem cells (hMSCs) with RGD-conjugated QDs during self replication and differentiation into osteogenic cell lineages.

  14. Label-free single cell analysis with a chip-based impedance flow cytometer

    Science.gov (United States)

    Pierzchalski, Arkadiusz; Hebeisen, Monika; Mittag, Anja; Di Berardino, Marco; Tarnok, Attila

    2010-02-01

    For description of cellular phenotypes and physiological states new developments are needed. Axetris' impedance flow cytometer (IFC) (Leister) is a new promising label-free alternative to fluorescence-based flow cytometry (FCM). IFC measures single cells at various frequencies simultaneously. The frequencies used for signal acquisition range from 0.1 to 20 MHz. The impedance signal provides information about cell volume (ionophore valinomycin. Changes in membrane potential were detectable at the level of cytoplasm conductivity (>4 MHz) and membrane capacitance (1-4 MHz). Our data indicate that IFC can be a valuable alternative to conventional FCM for various applications in the field of cell death and physiology. The work will be extended to address further potential applications of IFC in biotechnology and biomedical cell analysis, as well as in cell sorting.

  15. Metabolic labeling with (14C)-glucose of bloodstream and cell culture trypanosoma cruzi trypomastigotes:

    International Nuclear Information System (INIS)

    Trypomastigote forms of Trypanosoma cruzi from infected mouse blood and from cell culture were metabolically labeled by incubation with D-(14C)-glucose. Analysis by polyacrylamide gel electrophoresis of lysates from parasites of two strains (RA and CA1) showed a significantly different pattern. The difference was mainly quantitative when the blood and cell culture trypomastigotes of the RA strain were compared. Analysis of the culture medium by paper electrophoresis showed an anionic exometabolite only in the blood forms of both strains. (Author)

  16. A method for double-labeling sputum cells for p53 and cytokeratin

    Energy Technology Data Exchange (ETDEWEB)

    Neft, R.E.; Tierney, L.A.; Belinsky, S.A. [and others

    1995-12-01

    Molecular and immunological techniques may enhance the usefulness of sputum cytology as a screening tool for lung cancer. These techniques may also be useful in detecting and following the early progression of disease from metaplasia to dysplasia, carcinoma in situ, and finally to invasive carcinoma. Longitudinal information on the evolution of these malignant changes in the respiratory epithelium can be gained by prospective study of populations at high risk for lung cancer. This work is significant because double-labeling of cells in sputum with p53 and cytokeratin antibodies facilitates rapid screening of p53 positive neoplastic and preneoplastic lung cells by brightfield and fluorescence microscopy.

  17. Automatic labeling of molecular biomarkers on a cell-by-cell basis in immunohistochemistry images using convolutional neural networks

    Science.gov (United States)

    Sheikhzadeh, Fahime; Carraro, Anita; Korbelik, Jagoda; MacAulay, Calum; Guillaud, Martial; Ward, Rabab K.

    2016-03-01

    This paper addresses the problem of classifying cells expressing different biomarkers. A deep learning based method that can automatically localize and count the cells expressing each of the different biomarkers is proposed. To classify the cells, a Convolutional Neural Network (CNN) was employed. Images of Immunohistochemistry (IHC) stained slides that contain these cells were digitally scanned. The images were taken from digital scans of IHC stained cervical tissues, acquired for a clinical trial. More than 4,500 RGB images of cells were used to train the CNN. To evaluate our method, the cells were first manually labeled based on the expressing biomarkers. Then we performed the classification on 156 randomly selected images of cells that were not used in training the CNN. The accuracy of the classification was 92% in this preliminary data set. The results have shown that this method has a good potential in developing an automatic method for immunohistochemical analysis.

  18. A novel fluorescent label based on biological fluores-cent nanoparticles and its application in cell recognition

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Uniform-sized fluorescent nanoparticles have been prepared by employing silica as the shell and a highly luminescent dye complex of ruthenium ion and bipyridyl, tris(2,2′-bipyridyl) dichlororuthenium(Ⅱ) hexahydrate as the core of the nanoparticles. A novel fluorescent label method is proposed, which is based on the biological fluorescent nanoparticles on the foundation of nanotechnology, biotechnology and fluorescent label technology. In comparison with the conventional fluorophores as fluorescent labels such as fluorescein isothiocyanate (FITC) label, this new label shows more superiority in photochemical stability, detection sensitivity and application scope for the biomedicine research. SmIgG+ B lymphocytes isolated from the circulating blood of human beings can be easily recognized by using this new fluorescent label.

  19. Tc-99m Labeled HMPAO white Blood Cell Scintigraphy in Pediatric Patients

    Directory of Open Access Journals (Sweden)

    Funda Aydın

    2012-04-01

    Full Text Available Objective: 99mTc labeled hexamethylpropylene amine oxime (HMPAO white blood cell (WBC scintigraphy is a frequently used option for acute infection, particularly in pediatric patients. This scintigraphy is applied to detect sites of infection/inflammation in patients with fever of unknown origin, to find and follow up osteomyelitis, and to detect suspicion of acute appendicitis. The aim of this retrospective study was to evaluate the value of 99mTc-HMPAO labeled WBC scintigraphy in pediatric patients. Material and Methods: The study was conducted between January 2006 and December 2008 and included 13 patients (5 boys, 8 girls; mean age 6.9±6.2 years. Those patients who had suspicion of bone infection (n=7, fever of unknown origin (n=3, and suspicion of acute appendicitis (n=3 were evaluated retrospectively. 99mTc-HMPAO labeled WBC scintigraphy imaging was performed to all patients. Diagnosis was done according to operation and pathological results or clinical follow-up. Results: 99mTc-HMPAO labeled WBC scintigraphy has been found to be true positive in 6 cases, true negative in 6 cases, and false negative in one patient who had fewer unknown origin. The false negative case has been found to have encephalitis with MRI. Conclusion: Leukocyte scintigraphy has been described as a useful diagnostic tool in the diagnosis of suspicion of bone infection, fever of unknown origin and suspicion of acute appendicitis. 99mTc-HMPAO labeled WBC scintigraphy is a rapid and very accurate method for detecting those pathologies. Our results showed that WBC scintigraphy might be reliably used for diagnosis of suspected bone infection and acute appendicitis, fever of unknown origin, and acute appendicitis, in pediatric patient population. (MIRT 2012;21:13-18

  20. Initial evaluation of the use of USPIO cell labeling and noninvasive MR monitoring of human tissue-engineered vascular grafts in vivo

    OpenAIRE

    Nelson, G. N.; Roh, J. D.; Mirensky, T. L.; Y Wang; Yi, T.; Tellides, G.; Pober, J S; Shkarin, P.; Shapiro, E. M.; Saltzman, W M; Papademetris, X.; Fahmy, T. M.; Breuer, C.K.

    2008-01-01

    This pilot study examines noninvasive MR monitoring of tissue-engineered vascular grafts (TEVGs) in vivo using cells labeled with iron oxide nanoparticles. Human aortic smooth muscle cells (hASMCs) were labeled with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles. The labeled hASMCs, along with human aortic endothelial cells, were incorporated into eight TEVGs and were then surgically implanted as aortic interposition grafts in a C.B-17 SCID/bg mouse host. USPIO-labeled hASMCs p...

  1. Tunable coating of gold nanostars: tailoring robust SERS labels for cell imaging

    Science.gov (United States)

    Bassi, B.; Taglietti, A.; Galinetto, P.; Marchesi, N.; Pascale, A.; Cabrini, E.; Pallavicini, P.; Dacarro, G.

    2016-07-01

    Surface modification of noble metal nanoparticles with mixed molecular monolayers is one of the most powerful tools in nanotechnology, and is used to impart and tune new complex surface properties. In imaging techniques based on surface enhanced Raman spectroscopy (SERS), precise and controllable surface modifications are needed to carefully design reproducible, robust and adjustable SERS nanoprobes. We report here the attainment of SERS labels based on gold nanostars (GNSs) coated with a mixed monolayer composed of a poly ethylene glycol (PEG) thiol (neutral or negatively charged) that ensure stability in biological environments, and of a signalling unit 7-Mercapto-4-methylcoumarin as a Raman reporter molecule. The composition of the coating mixture is precisely controlled using an original method, allowing the modulation of the SERS intensity and ensuring overall nanoprobe stability. The further addition of a positively charged layer of poly (allylamine hydrocloride) on the surface of negatively charged SERS labels does not change the SERS response, but it promotes the penetration of GNSs in SH-SY5Y neuroblastoma cells. As an example of an application of such an approach, we demonstrate here the internalization of these new labels by means of visualization of cell morphology obtained with SERS mapping.

  2. Probing Xylan-Specific Raman Bands for Label-Free Imaging Xylan in Plant Cell Wall

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Yining; Yarbrough, John M.; Mittal, Ashutosh; Tucker, Melvin P.; Vinzant, Todd; Himmel, Michael E.

    2015-06-15

    Xylan constitutes a significant portion of biomass (e.g. 22% in corn stover used in this study). Xylan is also an important source of carbohydrates, besides cellulose, for renewable and sustainable energy applications. Currently used method for the localization of xylan in biomass is to use fluorescence confocal microscope to image the fluorescent dye labeled monoclonal antibody that specifically binds to xylan. With the rapid adoption of the Raman-based label-free chemical imaging techniques in biology, identifying Raman bands that are unique to xylan would be critical for the implementation of the above label-free techniques for in situ xylan imaging. Unlike lignin and cellulose that have long be assigned fingerprint Raman bands, specific Raman bands for xylan remain unclear. The major challenge is the cellulose in plant cell wall, which has chemical units highly similar to that of xylan. Here we report using xylanase to specifically remove xylan from feedstock. Under various degree of xylan removal, with minimum impact to other major cell wall components, i.e. lignin and cellulose, we have identified Raman bands that could be further tested for chemical imaging of xylan in biomass in situ.

  3. Efficient production of isotopically labeled proteins by cell-free synthesis: A practical protocol

    International Nuclear Information System (INIS)

    We provide detailed descriptions of our refined protocols for the cell-free production of labeled protein samples for NMR spectroscopy. These methods are efficient and overcome two critical problems associated with the use of conventional Escherichia coli extract systems. Endogenous amino acids normally present in E. coli S30 extracts dilute the added labeled amino acids and degrade the quality of NMR spectra of the target protein. This problem was solved by altering the protocol used in preparing the S30 extract so as to minimize the content of endogenous amino acids. The second problem encountered in conventional E. coli cell-free protein production is non-uniformity in the N-terminus of the target protein, which can complicate the NMR spectra. This problem was solved by adding a DNA sequence to the construct that codes for a cleavable N-terminal peptide tag. Addition of the tag serves to increase the yield of the protein as well as to ensure a homogeneous protein product following tag cleavage. We illustrate the method by describing its stepwise application to the production of calmodulin samples with different stable isotope labeling patterns for NMR analysis

  4. Synthesis of water-dispersible zinc oxide quantum dots with antibacterial activity and low cytotoxicity for cell labeling

    International Nuclear Information System (INIS)

    Typical photoluminescent semiconductor nanoparticles, called quantum dots (QDs), have potential applications in biological labeling. When used to label stem cells, QDs may impair the differentiation capacity of the stem cells. In this study, we synthesized zinc oxide (ZnO) QDs in methanol with an average size of ∼2 nm. We then employed two different types of polyethylene glycol (PEG) molecules (SH-PEG-NH2 and NH2-PEG-NH2) to conjugate ZnO QDs and made them water-dispersible. Fourier transform infrared spectroscopy spectra indicated the attachment of PEG molecules on ZnO QDs. No obvious size alteration was observed for ZnO QDs after PEG conjugation. The water-dispersible ZnO QDs still retained the antibacterial activity and fluorescence intensity. The cytotoxicity evaluation revealed that ZnO QDs at higher concentrations decreased cell viability but were generally safe at 30 ppm or below. Cell lines of hepatocytes (HepG2), osteoblasts (MC3T3-E1) and mesenchymal stem cells (MSCs) were successfully labeled by the water-dispersible ZnO QDs at 30 ppm. The ZnO QD-labeled MSCs maintained their stemness and differentiation capacity. Therefore, we conclude that the water-dispersible ZnO QDs developed in this study have antibacterial activity, low cytotoxicity, and proper labeling efficiency, and can be used to label a variety of cells including stem cells. (paper)

  5. Synthesis of water-dispersible zinc oxide quantum dots with antibacterial activity and low cytotoxicity for cell labeling

    Science.gov (United States)

    Hsu, Shan-hui; Lin, Ying Yi; Huang, Sherry; Lem, Kwok Wai; Huong Nguyen, Dinh; Lee, Dai Soo

    2013-11-01

    Typical photoluminescent semiconductor nanoparticles, called quantum dots (QDs), have potential applications in biological labeling. When used to label stem cells, QDs may impair the differentiation capacity of the stem cells. In this study, we synthesized zinc oxide (ZnO) QDs in methanol with an average size of ∼2 nm. We then employed two different types of polyethylene glycol (PEG) molecules (SH-PEG-NH2 and NH2-PEG-NH2) to conjugate ZnO QDs and made them water-dispersible. Fourier transform infrared spectroscopy spectra indicated the attachment of PEG molecules on ZnO QDs. No obvious size alteration was observed for ZnO QDs after PEG conjugation. The water-dispersible ZnO QDs still retained the antibacterial activity and fluorescence intensity. The cytotoxicity evaluation revealed that ZnO QDs at higher concentrations decreased cell viability but were generally safe at 30 ppm or below. Cell lines of hepatocytes (HepG2), osteoblasts (MC3T3-E1) and mesenchymal stem cells (MSCs) were successfully labeled by the water-dispersible ZnO QDs at 30 ppm. The ZnO QD-labeled MSCs maintained their stemness and differentiation capacity. Therefore, we conclude that the water-dispersible ZnO QDs developed in this study have antibacterial activity, low cytotoxicity, and proper labeling efficiency, and can be used to label a variety of cells including stem cells.

  6. In vivo transfer of intracellular labels from locally implanted bone marrow stromal cells to resident tissue macrophages.

    Directory of Open Access Journals (Sweden)

    Edyta Pawelczyk

    Full Text Available Intracellular labels such as dextran coated superparamagnetic iron oxide nanoparticles (SPION, bromodeoxyuridine (BrdU or green fluorescent protein (GFP are frequently used to study the fate of transplanted cells by in vivo magnetic resonance imaging or fluorescent microscopy. Bystander uptake of labeled cells by resident tissue macrophages (TM can confound the interpretation of the presence of intracellular labels especially during direct implantation of cells, which can result in more than 70% cell death. In this study we determined the percentages of TM that took up SPION, BrdU or GFP from labeled bone marrow stromal cells (BMSCs that were placed into areas of angiogenesis and inflammation in a mouse model known as Matrigel plaque perfusion assay. Cells recovered from digested plaques at various time points were analyzed by fluorescence microscopy and flow cytometry. The analysis of harvested plaques revealed 5% of BrdU(+, 5-10% of GFP(+ and 5-15% of dextran(+ macrophages. The transfer of the label was not dependent on cell dose or viability. Collectively, this study suggests that care should be taken to validate donor origin of cells using an independent marker by histology and to assess transplanted cells for TM markers prior to drawing conclusions about the in vivo behavior of transplanted cells.

  7. Tetrazine-Containing Amino Acid for Peptide Modification and Live Cell Labeling.

    Directory of Open Access Journals (Sweden)

    Zhongqiu Ni

    Full Text Available A novel amino acid derivative 3-(4-(1, 2, 4, 5-tetrazine-3-yl phenyl-2-aminopropanoic acid was synthesized in this study. The compound possessed better water-solubility and was synthesized more easily compared with the well-known and commercially available 3-(p-benzylamino-1, 2, 4, 5-tetrazine. Tetrazine-containing amino acid showed excellent stability in biological media and might be used for cancer cell labeling. Moreover, the compound remained relatively stable in 50% TFA/DCM with little decomposition after prolonged exposure at room temperature. The compound could be utilized as phenylalanine or tyrosine analogue in peptide modification, and the tetrazine-containing peptide demonstrated more significant biological activity than that of the parent peptide. The combination of tetrazine group and amino acid offered broad development prospects of the bioorthogonal labeling and peptide synthesis.

  8. Effect of HSA coated iron oxide labeling on human umbilical cord derived mesenchymal stem cells

    International Nuclear Information System (INIS)

    Human umbilical cord derived mesenchymal stem cells (hUC-MSCs) are known for self-renewal and differentiation into cells of various lineages like bone, cartilage and fat. They have been used in biomedical applications to treat degenerative disorders. However, to exploit the therapeutic potential of stem cells, there is a requirement of sensitive non-invasive imaging techniques which will offer the ability to track transplanted cells, bio-distribution, proliferation and differentiation. In this study, we have analyzed the efficacy of human serum albumin coated iron oxide nanoparticles (HSA-IONPs) on the differentiation of hUC-MSCs. The colloidal stability of the HSA-IONPs was tested over a long period of time (≥20 months) and the optimized concentration of HSA-IONPs for labeling the stem cells was 60 μg ml−1. Detailed in vitro assays have been performed to ascertain the effect of the nanoparticles (NPs) on stem cells. Lactate dehydrogenase (LDH) assay showed minimum release of LDH depicting the least disruptions in cellular membrane. At the same time, mitochondrial impairment of the cells was also not observed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Flow cytometry analysis revealed lesser generation of reactive oxygen species in HSA-IONPs labeled hUC-MSCs in comparison to bare and commercial IONPs. Transmission electron microscopy showed endocytic engulfment of the NPs by the hUC-MSCs. During the process, the gross morphologies of the actin cytoskeleton were found to be intact as shown by immunofluorescence microscopy. Also, the engulfment of the HSA-IONPs did not show any detrimental effect on the differentiation potential of the stem cells into adipocytes, osteocytes and chondrocytes, thereby confirming that the inherent properties of stem cells were maintained. (paper)

  9. Effect of HSA coated iron oxide labeling on human umbilical cord derived mesenchymal stem cells

    Science.gov (United States)

    Sanganeria, Purva; Chandra, Sudeshna; Bahadur, Dhirendra; Khanna, Aparna

    2015-03-01

    Human umbilical cord derived mesenchymal stem cells (hUC-MSCs) are known for self-renewal and differentiation into cells of various lineages like bone, cartilage and fat. They have been used in biomedical applications to treat degenerative disorders. However, to exploit the therapeutic potential of stem cells, there is a requirement of sensitive non-invasive imaging techniques which will offer the ability to track transplanted cells, bio-distribution, proliferation and differentiation. In this study, we have analyzed the efficacy of human serum albumin coated iron oxide nanoparticles (HSA-IONPs) on the differentiation of hUC-MSCs. The colloidal stability of the HSA-IONPs was tested over a long period of time (≥20 months) and the optimized concentration of HSA-IONPs for labeling the stem cells was 60 μg ml-1. Detailed in vitro assays have been performed to ascertain the effect of the nanoparticles (NPs) on stem cells. Lactate dehydrogenase (LDH) assay showed minimum release of LDH depicting the least disruptions in cellular membrane. At the same time, mitochondrial impairment of the cells was also not observed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Flow cytometry analysis revealed lesser generation of reactive oxygen species in HSA-IONPs labeled hUC-MSCs in comparison to bare and commercial IONPs. Transmission electron microscopy showed endocytic engulfment of the NPs by the hUC-MSCs. During the process, the gross morphologies of the actin cytoskeleton were found to be intact as shown by immunofluorescence microscopy. Also, the engulfment of the HSA-IONPs did not show any detrimental effect on the differentiation potential of the stem cells into adipocytes, osteocytes and chondrocytes, thereby confirming that the inherent properties of stem cells were maintained.

  10. International Society for Stem Cell Research

    Science.gov (United States)

    ... and Cell Therapy & ISSCR to Present Latest in Stem Cell Research, Gene Editing and Gene Therapy 18-21 Oct., ... share and discuss developments in the field of stem cell research and gene editing and therapies at the ESGCT/ ...

  11. Suicide of EMT-6 tumor cells by decays from radioactively-labelled sensitizer adducts

    International Nuclear Information System (INIS)

    Nitroaromatic radiosensitizers become metabolically bound preferentially to hypoxic cells and at least 10/sup 9/ adducts/cell can be tolerated as non-toxic. EMT-6 tumor cells have been incubated in hypoxia in the presence of /sup 3/H-Misonidazole and /sup 125/I-Azomycin Riboside for various times and the amount of /sup 3/H or /sup 125/I bound/cell was determined. Cells were stored as monolayers at 250C for up to 96 hr to accumulate radioactive decays and transferred at various times to 370C for colony-forming assays. No radiation inactivation was measured in cells which had incorporated at least 10/sup 6/ /sup 3/H or 10/sup 5/ /sup 125/I atoms. Previous studies had shown that -- 1% of MISO adducts to EMT-6 cells was associated with cellular DNA. These data indicate that the radiation-induced damage produced by these quantities of bound /sup 3/H or /sup 125/I causes little or not cell inactivation. The results of current studies to measure the colony-forming ability of sensitizer-labelled cells which have been stored in liquid nitrogen to facilitate the accumulation of more decays will be reported. These data suggest that a ''sensitizer-adduct suicide technique'' as a hypoxic cell selective adjunct to other cancer therapies is not feasible. These data are also instructive for those who attempt to develop radiolabelled ''tumor specific'' antibodies for therapeutic purposes

  12. Label-Free Imaging of Dynamic and Transient Calcium Signaling in Single Cells.

    Science.gov (United States)

    Lu, Jin; Li, Jinghong

    2015-11-01

    Cell signaling consists of diverse events that occur at various temporal and spatial scales, ranging from milliseconds to hours and from single biomolecules to cell populations. The pathway complexities require the development of new techniques that detect the overall signaling activities and are not limited to quantifying a single event. A plasmonic-based electrochemical impedance microscope (P-EIM) that can provide such data with excellent temporal and spatial resolution and does not require the addition of any labels for detection has now been developed. The highly dynamic and transient calcium signaling activities at the early stage of G-protein-coupled receptor (GPCR) stimulation were thus studied. It could be shown that a subpopulation of cells is more responsive towards agonist stimulation, and the heterogeneity of the local distributions and the transient activities of the ion channels during agonist-activated calcium flux in single HeLa cells were investigated.

  13. Inorganic phosphate nanorods are a novel fluorescent label in cell biology

    Directory of Open Access Journals (Sweden)

    Mukherjee Priyabrata

    2006-10-01

    Full Text Available Abstract We report the first use of inorganic fluorescent lanthanide (europium and terbium ortho phosphate [LnPO4·H2O, Ln = Eu and Tb] nanorods as a novel fluorescent label in cell biology. These nanorods, synthesized by the microwave technique, retain their fluorescent properties after internalization into human umbilical vein endothelial cells (HUVEC, 786-O cells, or renal carcinoma cells (RCC. The cellular internalization of these nanorods and their fluorescence properties were characterized by fluorescence spectroscopy (FS, differential interference contrast (DIC microscopy, confocal microscopy, and transmission electron microscopy (TEM. At concentrations up to 50 μg/ml, the use of [3H]-thymidine incorporation assays, apoptosis assays (TUNEL, and trypan blue exclusion illustrated the non-toxic nature of these nanorods, a major advantage over traditional organic dyes

  14. Transferrin protein nanospheres: a nanoplatform for receptor-mediated cancer cell labeling and gene delivery

    Science.gov (United States)

    McDonald, Michael A.; Spurlin, Tighe A.; Tona, Alessandro; Elliott, John T.; Halter, Michael; Plant, Anne L.

    2010-02-01

    This paper presents preliminary results on the use of transferrin protein nanospheres (TfpNS) for targeting cancer cells in vitro. Protein nanospheres represent an easily prepared and modifiable nanoplatform for receptor-specific targeting, molecular imaging and gene delivery. Rhodamine B isothiocyanate conjugated TfpNS (RBITC-TfpNS) show significantly enhanced uptake in vitro in SK-MEL-28 human malignant melanoma cells known to overexpress transferrin receptors compared to controls. RBITCTfpNS labeling of the cancer cells is due to transferrin receptor-mediated uptake, as demonstrated by competitive inhibition with native transferrin. Initial fluorescence microscopy studies indicate GFP plasmid can be transfected into melanoma cells via GFP plasmid encapsulated by TfpNS.

  15. Label Free Detection of CD4+ and CD8+ T Cells Using the Optofluidic Ring Resonator

    Directory of Open Access Journals (Sweden)

    John T. Gohring

    2010-06-01

    Full Text Available We have demonstrated label free detection of CD4+ and CD8+ T-Lymphocyte whole cells and CD4+ T-Lymphocyte cell lysis using the optofluidic ring resonator (OFRR sensor. The OFRR sensing platform incorporates microfluidics and photonics in a setup that utilizes small sample volume and achieves a fast detection time. In this work, white blood cells were isolated from healthy blood and the concentrations were adjusted to match T-Lymphocyte levels of individuals infected with HIV. Detection was accomplished by immobilizing CD4 and CD8 antibodies on the inner surface of the OFRR. Sensing results show excellent detection of CD4+ and CD8+ T-Lymphocyte cells at medically significant concentrations with a detection time of approximately 30 minutes. This work will lead to a rapid and low-cost sensing device that can provide a CD4 and CD8 count as a measure of HIV progression.

  16. Human induced pluripotent stem cells labeled with fluorescent magnetic nanoparticles for targeted imaging and hyperthermia therapy for gastric cancer

    International Nuclear Information System (INIS)

    Human induced pluripotent stem (iPS) cells exhibit great potential for generating functional human cells for medical therapies. In this paper, we report for use of human iPS cells labeled with fluorescent magnetic nanoparticles (FMNPs) for targeted imaging and synergistic therapy of gastric cancer cells in vivo. Human iPS cells were prepared and cultured for 72 h. The culture medium was collected, and then was co-incubated with MGC803 cells. Cell viability was analyzed by the MTT method. FMNP-labeled human iPS cells were prepared and injected into gastric cancer-bearing nude mice. The mouse model was observed using a small-animal imaging system. The nude mice were irradiated under an external alternating magnetic field and evaluated using an infrared thermal mapping instrument. Tumor sizes were measured weekly. iPS cells and the collected culture medium inhibited the growth of MGC803 cells. FMNP-labeled human iPS cells targeted and imaged gastric cancer cells in vivo, as well as inhibited cancer growth in vivo through the external magnetic field. FMNP-labeled human iPS cells exhibit considerable potential in applications such as targeted dual-mode imaging and synergistic therapy for early gastric cancer

  17. The Non-Specific Binding of Fluorescent-Labeled MiRNAs on Cell Surface by Hydrophobic Interaction.

    Directory of Open Access Journals (Sweden)

    Ting Lu

    Full Text Available MicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. The fluorescent labeling and lipofection are two common methods for changing the levels and locating the position of cellular miRNAs. Despite many studies about the mechanism of DNA/RNA lipofection, little is known about the characteristics, mechanisms and specificity of lipofection of fluorescent-labeled miRNAs.Therefore, miRNAs labeled with different fluorescent dyes were transfected into adherent and suspension cells using lipofection reagent. Then, the non-specific binding and its mechanism were investigated by flow cytometer and laser confocal microscopy. The results showed that miRNAs labeled with Cy5 (cyanine fluorescent dye could firmly bind to the surface of adherent cells (Hela and suspended cells (K562 even without lipofection reagent. The binding of miRNAs labeled with FAM (carboxyl fluorescein to K562 cells was obvious, but it was not significant in Hela cells. After lipofectamine reagent was added, most of the fluorescently labeled miRNAs binding to the surface of Hela cells were transfected into intra-cell because of the high transfection efficiency, however, most of them were still binding to the surface of K562 cells. Moreover, the high-salt buffer which could destroy the electrostatic interactions did not affect the above-mentioned non-specific binding, but the organic solvent which could destroy the hydrophobic interactions eliminated it.These results implied that the fluorescent-labeled miRNAs could non-specifically bind to the cell surface by hydrophobic interaction. It would lead to significant errors in the estimation of transfection efficiency only according to the cellular fluorescence intensity. Therefore, other methods to evaluate the transfection efficiency and more appropriate fluorescent dyes should be used according to the cell types for the accuracy of results.

  18. Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells

    Science.gov (United States)

    Hofemeier, Arne D.; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F. W.; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

    2016-05-01

    Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO43‑ symmetric stretch vibrations at 959 cm‑1 assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue–implant-interfaces or disease diagnosis.

  19. Quantifying size-dependent interactions between fluorescently labeled polystyrene nanoparticles and mammalian cells

    Science.gov (United States)

    2012-01-01

    Background Nanoparticles (NPs) are currently used in a wide variety of fields such as technology, medicine and industry. Due to the novelty of these applications and to ensure their success, a precise characterization of the interactions between NPs and cells is essential. Findings The current study explores the uptake of polystyrene NPs by 1321N1 human astrocytoma and A549 human lung carcinoma cell lines. In this work we show for the first time a comparison of the uptake rates of fluorescently labeled carboxylated polystyrene (PS) NPs of different sizes (20, 40 and 100 nm) in two different cell types, keeping the number of NPs per unit volume constant for all sizes. We propose a reliable methodology to control the dose of fluorescently labeled NPs, by counting individual NPs using automated particle detection from 3D confocal microscopy images. The possibility of detecting individual NPs also allowed us to calculate the size of each nanoparticle and compare the fluorescence of single NPs across different sizes, thereby providing a robust platform for normalization of NP internalization experiments as measured by flow cytometry. Conclusions Our findings show that 40 nm NPs are internalized faster than 20 nm or 100 nm particles in both cell lines studied, suggesting that there is a privileged size gap in which the internalization of NPs is higher. PMID:23006133

  20. Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells

    Science.gov (United States)

    Hofemeier, Arne D.; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F. W.; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

    2016-05-01

    Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO43- symmetric stretch vibrations at 959 cm-1 assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue-implant-interfaces or disease diagnosis.

  1. Quantifying size-dependent interactions between fluorescently labeled polystyrene nanoparticles and mammalian cells

    Directory of Open Access Journals (Sweden)

    Varela Juan A

    2012-09-01

    Full Text Available Abstract Background Nanoparticles (NPs are currently used in a wide variety of fields such as technology, medicine and industry. Due to the novelty of these applications and to ensure their success, a precise characterization of the interactions between NPs and cells is essential. Findings The current study explores the uptake of polystyrene NPs by 1321N1 human astrocytoma and A549 human lung carcinoma cell lines. In this work we show for the first time a comparison of the uptake rates of fluorescently labeled carboxylated polystyrene (PS NPs of different sizes (20, 40 and 100 nm in two different cell types, keeping the number of NPs per unit volume constant for all sizes. We propose a reliable methodology to control the dose of fluorescently labeled NPs, by counting individual NPs using automated particle detection from 3D confocal microscopy images. The possibility of detecting individual NPs also allowed us to calculate the size of each nanoparticle and compare the fluorescence of single NPs across different sizes, thereby providing a robust platform for normalization of NP internalization experiments as measured by flow cytometry. Conclusions Our findings show that 40 nm NPs are internalized faster than 20 nm or 100 nm particles in both cell lines studied, suggesting that there is a privileged size gap in which the internalization of NPs is higher.

  2. A review of European research on consumer response to nutrition information on food labels

    DEFF Research Database (Denmark)

    Grunert, Klaus G.; Wills, Josephine M.

    2007-01-01

    The aim of this study was to review research conducted in 2003-2006 in the EU-15 countries on how consumers perceive, understand, like and use nutrition information on food labels. Based on a search of databases on academic publications, Google-based search, and enquiries directed to a range...... in order to identify key findings and common themes across the studies. The studies show widespread consumer interest in nutrition information on food packages, though this interest varies across situations and products. Consumers like the idea of simplified front of pack information but differ...... of food retailers, food companies, consumer associations and government agencies, a total of 58 studies were identified. These studies were summarised using a standard format guided by a model of consumer information processing, and these summaries were subsequently processed using the MAXqda software...

  3. Report of the research co-ordination meeting on labelling techniques of biomolecules for targeted radiotherapy

    International Nuclear Information System (INIS)

    The CRP's focus is on the preparation of site-specific radiopharmaceuticals labelled with with β- emitters for the treatment of cancer. The radiopharmaceuticals should be designed in such a way to deliver the therapeutic doses with high specificity to tumour sites and with minimum dose to other organs. The 2nd Research Co-ordination Meeting (RCM) took place in Mumbai, India from 31 January to 4 February 2000. The present report includes the results of all the participants including the report of the participant from Pakistan who could not attend the meeting. During the second RCM, three main aspects were dealt with viz. the different therapeutic isotopes studied, different carrier molecules like peptides and antibody and the biological aspects of these preparations

  4. Report of the research co-ordination meeting on labelling techniques of biomolecules for targeted radiotherapy

    International Nuclear Information System (INIS)

    The CRP's focus is on the preparation of site-specific radiopharmaceuticals labelled with β-emitters for the treatment of cancer. The radiopharmaceuticals should be designed in such a way to deliver the therapeutic doses with high specificity to tumour sites and with minimum dose to other organs. The 2. Research Co-ordination Meeting (RCM) took place in Mumbai, India from 31 January to 4 February 2000. The present report includes the results of all the participants including the report of the participant from Pakistan who could not attend the meeting. During the second RCM, three main aspects were dealt with viz. the different therapeutic isotopes studied, different carrier molecules like peptides and antibody and the biological aspects of these preparations

  5. Relation between clinical mature and immature lymphocyte cells in human peripheral blood and their spatial label free scattering patterns

    Science.gov (United States)

    Zhang, Lu; Zhao, Xin; Zhang, Zhenxi; Zhao, Hong; Chen, Wei; Yuan, Li

    2016-07-01

    A single living cell's light scattering pattern (LSP) in the horizontal plane, which has been denoted as the cell's "2D fingerprint," may provide a powerful label-free detection tool in clinical applications. We have recently studied the LSP in spatial scattering planes, denoted as the cell's "3D fingerprint," for mature and immature lymphocyte cells in human peripheral blood. The effects of membrane size, morphology, and the existence of the nucleus on the spatial LSP are discussed. In order to distinguish clinical label-free mature and immature lymphocytes, the special features of the spatial LSP are studied by statistical method in both the spatial and frequency domains. Spatial LSP provides rich information on the cell's morphology and contents, which can distinguish mature from immature lymphocyte cells and hence ultimately it may be a useful label-free technique for clinical leukemia diagnosis.

  6. Dose dependent side effect of superparamagnetic iron oxide nanoparticle labeling on cell motility in two fetal stem cell populations.

    Directory of Open Access Journals (Sweden)

    Valentina Diana

    Full Text Available Multipotent stem cells (SCs could substitute damaged cells and also rescue degeneration through the secretion of trophic factors able to activate the endogenous SC compartment. Therefore, fetal SCs, characterized by high proliferation rate and devoid of ethical concern, appear promising candidate, particularly for the treatment of neurodegenerative diseases. Super Paramagnetic Iron Oxide nanoparticles (SPIOn, routinely used for pre-clinical cell imaging and already approved for clinical practice, allow tracking of transplanted SCs and characterization of their fate within the host tissue, when combined with Magnetic Resonance Imaging (MRI. In this work we investigated how SPIOn could influence cell migration after internalization in two fetal SC populations: human amniotic fluid and chorial villi SCs were labeled with SPIOn and their motility was evaluated. We found that SPIOn loading significantly reduced SC movements without increasing production of Reactive Oxygen Species (ROS. Moreover, motility impairment was directly proportional to the amount of loaded SPIOn while a chemoattractant-induced recovery was obtained by increasing serum levels. Interestingly, the migration rate of SPIOn labeled cells was also significantly influenced by a degenerative surrounding. In conclusion, this work highlights how SPIOn labeling affects SC motility in vitro in a dose-dependent manner, shedding the light on an important parameter for the creation of clinical protocols. Establishment of an optimal SPIOn dose that enables both a good visualization of grafted cells by MRI and the physiological migration rate is a main step in order to maximize the effects of SC therapy in both animal models of neurodegeneration and clinical studies.

  7. BrdU-label-retaining cells in rat eccrine sweat glands over time.

    Science.gov (United States)

    Li, Haihong; Zhang, Mingjun; Li, Xuexue; Chen, Lu; Zhang, Bingna; Tang, Shijie; Fu, Xiaobing

    2016-03-01

    Cell proliferation and turnover are fueled by stem cells. In a previous study, we demonstrated that rat eccrine sweat glands contained abundant bromodeoxyuridine (BrdU)-label-retaining cells (LRCs). However, morphological observations showed that eccrine sweat glands usually show little or no signs of homeostatic change. In this study, we account for why the homeostatic change is rare in eccrine sweat glands based on cytokinetic changes in BrdU-LRC turnover, and also determine the BrdU-labeled cell type. Thirty-six newborn SD rats, were injected intraperitoneally with 50mg/kg BrdU twice daily at a 2h interval for 4 consecutive days. After a chase period of 4, 6, 8, 12, 24 and 32 weeks, rats were euthanized, and the hind footpads were removed and processed for BrdU immunostaining, and BrdU/α-SMA and BrdU/K14 double-immunostaining. BrdU-LRCs were observed in the ducts, secretory coils and mesenchymal cells at all survival time points. The percentage of BrdU(+) cells in rat eccrine sweat glands averaged 4.2±1.2% after 4 weeks of chase, increased slightly by the 6th week, averaging 4.4±0.9%, and peaked at 8 weeks, averaging 5.3±1.0%. Subsequently, the average percentage of BrdU(+) cells declined to 3.2±0.8% by the 32nd week. There was no difference in the percentage of BrdU-LRCs among the different survival time points except that a significant difference in the percentage of BrdU-LRCs detected at 24 weeks versus 8 weeks, and 32 weeks versus 8 weeks, was observed. We concluded that the BrdU-LRCs turnover is slow in eccrine sweat glands. PMID:26657518

  8. [Rapidly labelled low molecular weight components in nucleic acid preparations from plant cells].

    Science.gov (United States)

    Richter, G; Grotha, R

    1974-09-01

    After pulse-labelling with [(3)H]nucleosides and [(3)H]orotic acid of freely suspended callus cells of Petroselinum sativum and tissue fragments of the liverwort Riella helicophylla, rapidly labelled low molecular weight components were detected among the total nucleic acids when these were extracted in the presence of Mg(2+) and finally precipitated with alcohol. These highly labelled species could clearly be distinguished from the 5 S- and 4 S-RNA on the basis of their migration in agarose-polyacrylamide gels (2.4%) and their elution from Sephadex G-150 columns. No degradation was obtained with DNase and RNase. By using [(14)C]ATP as a marker it was found that the low molecular components consisted mainly of nucleoside triphosphates. Only small amounts of nucleoside diphosphates were detected, which were obviously formed by degradation of the former. Nucleic acid preparations free of nucleoside phosphates were obtained by using Mg-free extraction buffers containing EDTA. PMID:24458196

  9. Cell Wall Growth and Modulation Dynamics in a Model Unicellular Green Alga—Penium margaritaceum: Live Cell Labeling with Monoclonal Antibodies

    OpenAIRE

    Domozych, David S; Hannah Brechka; Alicia Britton; Marc Toso

    2011-01-01

    Penium margaritaceum is a unicellular charophycean green alga that possesses cell wall polymers similar to those of land plants. Several wall macromolecules of this alga are recognized by monoclonal antibodies specific for wall polymer epitopes of land plants. Immunofluorescence protocols using these antibodies may be employed to label specific cell wall constituents of live cells. Fluorescent labeling persists for several days, and this attribute allows for tracing of wall epitopes in both l...

  10. Tracking of [18F]FDG-labeled natural killer cells to HER2/neu-positive tumors

    International Nuclear Information System (INIS)

    Introduction: The objective of this study was to label the human natural killer (NK) cell line NK-92 with [18F]fluoro-deoxy-glucose (FDG) for subsequent in vivo tracking to HER2/neu-positive tumors. Methods: NK-92 cells were genetically modified to NK-92-scFv(FRP5)-zeta cells, which express a chimeric antigen receptor that is specific to the tumor-associated ErbB2 (HER2/neu) antigen. NK-92 and NK-92-scFv(FRP5)-zeta cells were labeled with [18F]FDG by simple incubation at different settings. Labeling efficiency was evaluated by a gamma counter. Subsequently, [18F]FDG-labeled parental NK-92 or NK-92-scFv(FRP5)-zeta cells were intravenously injected into mice with implanted HER2/neu-positive NIH/3T3 tumors. Radioactivity in tumors was quantified by digital autoradiography and correlated with histopathology. Results: The NK-92 and NK-92-scFv(FRP5)-zeta cells could be efficiently labeled with [18F]FDG by simple incubation. Optimal labeling efficiencies (80%) were achieved using an incubation period of 60 min and additional insulin (10 IU/ml). After injection of 5x106 [18F]FDG-labeled NK-92-scFv(FRP5)-zeta cells into tumor-bearing mice, digital autoradiography showed an increased uptake of radioactivity in HER2/neu-positive tumors at 60 min postinjection. Conversely, injection of 5x106 NK-92 cells not directed against HER2/neu receptors did not result in increased uptake of radioactivity in the tumors. Histopathology confirmed an accumulation of the NK-92-scFv(FRP5)-zeta cells, but not the parental NK cells, in tumor tissues. Conclusion: The human NK cell line NK-92 can be directed against HER2/neu antigens by genetic modification. The genetically modified NK cells can be efficiently labeled with [18F]FDG, and the accumulation of these labeled NK cells in HER2/neu-positive tumors can be monitored with autoradiography

  11. Human induced pluripotent stem cells labeled with lfuorescent magnetic nanoparticles for targeted imaging and hyperthermia therapy for gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Chao Li; Wei-Lin Jin; Da-Xiang Cui; Jing Ruan; Meng Yang; Fei Pan; Guo Gao; Su Qu; You-Lan Shen; Yong-Jun Dang; Kan Wang

    2015-01-01

    Objective: Human induced pluripotent stem (iPS) cells exhibit great potential for generating functional human cells for medical therapies. In this paper, we report for use of human iPS cells labeled with lfuorescent magnetic nanoparticles (FMNPs) for targeted imaging and synergistic therapy of gastric cancer cellsin vivo. Methods: Human iPS cells were prepared and cultured for 72 h. The culture medium was collected, and then was co-incubated with MGC803 cells. Cell viability was analyzed by the MTT method. FMNP-labeled human iPS cells were prepared and injected into gastric cancer-bearing nude mice. hTe mouse model was observed using a small-animal imaging system. hTe nude mice were irradiated under an external alternating magnetic ifeld and evaluated using an infrared thermal mapping instrument. Tumor sizes were measured weekly. Results: iPS cells and the collected culture medium inhibited the growth of MGC803 cells. FMNP-labeled human iPS cells targeted and imaged gastric cancer cellsin vivo, as well as inhibited cancer growthin vivo through the external magnetic ifeld. Conclusion: FMNP-labeled human iPS cells exhibit considerable potential in applications such as targeted dual-mode imaging and synergistic therapy for early gastric cancer.

  12. Incorporation and Degradation of 14C and 3H-labeled Thymidine by Sugarcane Cells in Suspension Culture 12

    Science.gov (United States)

    Lesley, Stanley M.; Maretzki, Andrew; Nickell, Louis G.

    1980-01-01

    Sugarcane cells growing in suspension culture degrade exogenous thymidine, releasing thymine. Thymine is not utilized for DNA synthesis. Thymine is rapidly catabolized to β-aminoisobutyric acid which is found within the cell. Thymidine in the medium is used for DNA synthesis. The label of [2-14C]thymidine is lost as 14CO2, but the label of [3H]methylthymidine is found in the cell as [3H]β-aminoisobutyric acid, some of which is used for the synthesis of other cell components. The degradation of thymidine can be partially inhibited by addition of certain substituted pyrimidines. PMID:16661365

  13. In vivo tracking of stem cells labeled with a nanoparticle in Alzheimer's disease animal model

    Science.gov (United States)

    Ha, Sungji; Suh, Yoo-Hun; Chang, Keun-A.

    2013-05-01

    Stem cell therapy is a promising tool for the treatment of diverse conditions including neurodegenerative diseases. To understand transplanted stem cell biology, in vivo imaging is necessary. Nano material has great potential for in vivo imaging and several noninvasive methods are used such as magnetic resonance imaging (MRI), positron emission tomography (PET), Fluorescence imaging (FI) and Near-infrared fluorescence imaging (NIRFI). However, each method has limitations for in vivo imaging. To overcome these limitations, multimodal nanoprobes have been developed. In the present study, we intravenously injected human adipose derived stem cells (hASCs) that labeled with multimodal nano particle, LEO-LIVETM-Magnoxide 797 or 675, into the Tg2576 mice, Alzheimer's disease (AD) mouse model. Sequential in vivo tracking was performed with mice injected with hASCs. We could found fluorescence signals until 10 days after injection.

  14. Label-free Imaging of Arterial Cells and Extracellular Matrix Using a Multimodal CARS Microscope.

    Science.gov (United States)

    Wang, Han-Wei; Le, Thuc T; Cheng, Ji-Xin

    2008-04-01

    A multimodal nonlinear optical imaging system that integrates coherent anti-Stokes Raman scattering (CARS), sum-frequency generation (SFG), and two-photon excitation fluorescence (TPEF) on the same platform was developed and applied to visualize single cells and extracellular matrix in fresh carotid arteries. CARS signals arising from CH(2)-rich membranes allowed visualization of endothelial cells and smooth muscle cells of the arterial wall. Additionally, CARS microscopy allowed vibrational imaging of elastin and collagen fibrils which are also rich in CH(2) bonds. The extracellular matrix organization were further confirmed by TPEF signals arising from elastin's autofluorescence and SFG signals arising from collagen fibrils' non-centrosymmetric structure. Label-free imaging of significant components of arterial tissues suggests the potential application of multimodal nonlinear optical microscopy to monitor onset and progression of arterial diseases.

  15. Nanosensors for label-free measurement of sodium ion fluxes of neuronal cells

    Energy Technology Data Exchange (ETDEWEB)

    Gebinoga, Michael, E-mail: michael.gebinoga@tu-ilmenau.de [ZIK MacroNano Microfluidics and Biosensors, Technical University Ilmenau, P.O. Box 100565, D-98684 Ilmenau (Germany); Silveira, Liele; Cimalla, Irina [ZIK MacroNano Microfluidics and Biosensors, Technical University Ilmenau, P.O. Box 100565, D-98684 Ilmenau (Germany); Dumitrescu, Andreea [University of Pennsylvania - School of Engineering and Applied Science, Philadelphia, PA 19104-6391 (United States); Kittler, Mario; Luebbers, Benedikt; Becker, Annette [ZIK MacroNano Microfluidics and Biosensors, Technical University Ilmenau, P.O. Box 100565, D-98684 Ilmenau (Germany); Lebedev, Vadim [Fraunhofer Institute for Solid State Physics, Tullastr. 7, D-79108 Freiburg (Germany); Schober, Andreas [ZIK MacroNano Microfluidics and Biosensors, Technical University Ilmenau, P.O. Box 100565, D-98684 Ilmenau (Germany)

    2010-05-25

    Novel nanosensors based on aluminium gallium nitrides (AlGaN/GaN) high electron mobility transistors have been of high interest during the last years, especially for their electrical characteristics as open gate field effect transistors. These nanosensors provide a valuable tool for high content screening in drug discovery, cell monitoring and liquid analyses focusing on applications of electrochemical detection technology. Our own measurements with these sensors confirm their pH sensitivity and in addition the possibility of detection of other ions in aqueous media. These measurements deal with the reactions of NG 108-15 (mouse neuroblastoma x rat glioma hybrid) neuronal cells in response to different acetylcholinesterase inhibitors. Our experimental approach shows some advantages. The first advantage is the label-free measurement of ion fluxes, and another advantage is the possibility non-destructively to estimate cell signals.

  16. Quantitative Proteomics: Measuring Protein Synthesis Using 15N Amino Acids Labeling in Pancreas Cancer Cells

    Science.gov (United States)

    Zhao, Yingchun; Lee, Wai-Nang Paul; Lim, Shu; Go, Vay Liang; Xiao, Jing; Cao, Rui; Zhang, Hengwei; Recker, Robert; Xiao, Gary Guishan

    2010-01-01

    Pancreatic cancer MIA PaCa cells were cultured in the presence and absence of 15N amino acids mixture for 72 hours. During protein synthesis, the incorporation of 15N amino acids results in a new mass isotopomer distribution in protein, which is approximated by the concatenation of two binomial distributions of 13C and 15N. Fraction of protein synthesis (FSR) can thus be determined from the relative intensities of the ‘labeled’ (new) and the ‘unlabeled” (old) spectra. Six prominent spots were picked from 2-D gels of proteins from lysates of cells cultured in 0% (control), and 50% and 33% 15N enriched media. These protein spots were digested and analyzed with MALDI-TOF/TOF. The isotopomer distribution of peptides after labeling can be fully accounted for by the labeled (new) and unlabeled (old) peptides. The ratio of the new and old peptide fractions was determined using multiple regression analysis of the observed spectrum as a linear combination of the expected new and the old spectra. The fractional protein synthesis rates calculated from such ratios of same peptide from cells grown in 50% and 33% 15N amino acid enrichments were comparable to each other. The FSR of these six identified proteins ranged between 44–76%. PMID:19072287

  17. Indium-111-oxine labeled leukocyte uptake in Ki-1-positive anaplastic large cell lymphoma

    Energy Technology Data Exchange (ETDEWEB)

    Chiu, W.; Amodio, J.B.; Scharf, S.C. (New York Univ., NY (United States). Dept. of Radiology); Rivlin, K.A. (Department of Hematology and Oncology, New York Univ. Medical Center, NY (United States)); Desai, P. (Department of Pathology, Hospital for Joint Diseases-Orthopaedic Inst., New York, NY (United States)); Breuer, F. (Department of Pathology, Lenox Hill Hospital, New York, NY (United States))

    1999-08-01

    Indium-111-oxine labeled leukocyte ([sup 111]In-WBC) scintigraphy is well known for its ability to localize in areas of active infection, but not in areas of lymphomatous involvement. We present a case of Ki-1-positive anaplastic large-cell lymphoma that was initially thought to be a case of multifocal osteomyelitis because of positive uptake on a [sup 111]In-WBC scan. The areas of abnormal uptake on the indium scan were demonstrated histopathologically to be sites of lymphomatous involvement in bone. (orig.) With 4 figs., 3 refs.

  18. Transplantation of magnetically labeled mesenchymal stem cells improves cardiac function in a swine myocardial infarction model

    Institute of Scientific and Technical Information of China (English)

    QI Chun-mei; JU Sheng-hong; MA Ming; TANG Yao-liang; MA Gen-shan; LIU Nai-feng; SHEN Cheng-xing; CHEN Zhong; LIU Xiao-jun; HU Yao-peng; ZHANG Xiao-li; TENG Gao-jun

    2008-01-01

    Background Mesenchymal stem cells (MSCs) transplantation provides a new approach for myocardial repair.However,many important fundamental questions about MSCs transplantation remain unanswered.There is an urgent need to identify MSCs from the beating heart and analyze the efficacy of this new approach.This study aimed to localize the magnetically labeled MSCs(MR-MSCs)and monitor the restorative effects of MR-MSCs with magnetic resonance(MR) imaging.Methods Acute myocardial infarction(AMI)was created in swine by a balloon occlusion of the left anterior descending coronary artery.Cells were delivered via intracoronary infusion after myocardial infarction.Infarct size change and cardiac function were assessed with 3.0T MR scanner.The results were then confirmed by histological and western blot analysis.All statistical procedures were performed with Systat (SPSS version 12.01).Results A total of 26 swine were divided into four groups(sham-operated group,n=6;AMI group with PBS transplantation,n=6;labeled MSCs group,n=7;unlabeled MSCs group,n=7).MSCs,MR-MSCs(107 cells)or PBS were delivered by intracoronary injection after MI and serial cardiac MR imaging studies were performed at 0,4 and 8 weeks after transplantation.MR imaging demonstrated MI size decreased after MSCs transplantation in labeled and unlabeled groups,however,increases were seen in the AMI group at 8 weeks after MI.The left ventricular eiection fraction(LVEF) was slightly increased in the AMI group((41.87±2.45)%vs(39.04±2.80)%,P>0.05),but significantly improved in the MR-MSCs group((56.85±1.29)%vs(40.67±2.00)%,P<0.05)and unlabeled group((55.38±1.07)%vs(41.78±2.08)%,P<0.05) at 8 weeks after treatment.MR-MSCs were further confirmed by Prussian blue and immunofluorescent staining.Western blot analvsis demonstrated that there was an increased expression of cardiomyocyte markers such as myosin heavy chain and troponin T in the MSCs treatment groups and the ratio of matrix metalloproteinase 2 to

  19. Medical applications of nanoparticles in biological imaging, cell labeling, antimicrobial agents, and anticancer nanodrugs.

    Science.gov (United States)

    Singh, Ravina; Nalwa, Hari Singh

    2011-08-01

    This article reviews the applications of nanotechnology in the fields of medical and life sciences. Nanoparticles have shown promising applications from diagnosis to treatment of various types of diseases including cancer. In this review, we discuss the applications of nanostructured materials such as nanoparticles, quantum dots, nanorods, nanowires, and carbon nanotubes in diagnostics, biomarkers, cell labeling, contrast agents for biological imaging, antimicrobial agents, drug delivery systems, and anticancer nanodrugs for treatment of cancer and other infectious diseases. The adverse affects of nanoparticles on human skin from daily use in cosmetics and general toxicology of nanoscale materials are also reviewed. PMID:21870454

  20. The nematode stoma: Homology of cell architecture with improved understanding by confocal microscopy of labeled cell boundaries.

    Science.gov (United States)

    Jay Burr, A H; Baldwin, James G

    2016-09-01

    Nematode stomas vary widely in the cuticular structures evolved for different feeding strategies, yet the arrangement of the epithelial cell classes that form these structures may be conserved. This article addresses several issues that have impeded the full acceptance of this hypothesis including controversies arising from the structure of the Caenorhabditis elegans stoma. We investigated fluorescent antibody labeling of cell boundaries in conjunction with confocal microscopy as an alternative to transmission electron microscopy (TEM), using MH27 to label apical junctions in C. elegans and two other species. Accurately spaced optical sections collected by the confocal microscope provide a three-dimensional array of pixels (voxels) that, using image-processing software, can be rotated and sectioned at accurately chosen thicknesses and locations. Ribbons of fluorescence clearly identify cell boundaries along the luminal cuticle in C. elegans and Zeldia punctata and less clearly in Bunonema sp. The patterns render cell classes and their relationships readily identifiable. In the C. elegans stoma they correct a misreading of serial TEMs that was not congruent with architecture in other nematodes-the row of marginal cells is now seen to be continuous as in other nematodes, rather than being interrupted by encircling pm1 cells. Also impeding understanding, the reference to certain cell classes as 'epithelial' and others as "muscle" in the C. elegans literature is at variance with muscle expression in most other taxa. For consistent comparison among species, we propose that these cell class descriptors based on function be replaced by topological terms. With these and other confusing concepts and terminology removed, the homology of the cellular architecture among taxa becomes obvious. We provide a corrected description of the cell architecture of the C. elegans stoma and examples of how it is modified in other taxa with different feeding strategies. J. Morphol. 277

  1. The nematode stoma: Homology of cell architecture with improved understanding by confocal microscopy of labeled cell boundaries.

    Science.gov (United States)

    Jay Burr, A H; Baldwin, James G

    2016-09-01

    Nematode stomas vary widely in the cuticular structures evolved for different feeding strategies, yet the arrangement of the epithelial cell classes that form these structures may be conserved. This article addresses several issues that have impeded the full acceptance of this hypothesis including controversies arising from the structure of the Caenorhabditis elegans stoma. We investigated fluorescent antibody labeling of cell boundaries in conjunction with confocal microscopy as an alternative to transmission electron microscopy (TEM), using MH27 to label apical junctions in C. elegans and two other species. Accurately spaced optical sections collected by the confocal microscope provide a three-dimensional array of pixels (voxels) that, using image-processing software, can be rotated and sectioned at accurately chosen thicknesses and locations. Ribbons of fluorescence clearly identify cell boundaries along the luminal cuticle in C. elegans and Zeldia punctata and less clearly in Bunonema sp. The patterns render cell classes and their relationships readily identifiable. In the C. elegans stoma they correct a misreading of serial TEMs that was not congruent with architecture in other nematodes-the row of marginal cells is now seen to be continuous as in other nematodes, rather than being interrupted by encircling pm1 cells. Also impeding understanding, the reference to certain cell classes as 'epithelial' and others as "muscle" in the C. elegans literature is at variance with muscle expression in most other taxa. For consistent comparison among species, we propose that these cell class descriptors based on function be replaced by topological terms. With these and other confusing concepts and terminology removed, the homology of the cellular architecture among taxa becomes obvious. We provide a corrected description of the cell architecture of the C. elegans stoma and examples of how it is modified in other taxa with different feeding strategies. J. Morphol. 277

  2. In vivo MR imaging of nanometer magnetically labeled bone marrow stromal cells transplanted via portal vein in rat liver

    International Nuclear Information System (INIS)

    Objective: To evaluate in vivo magnetic resonance imaging with a conventional 1.5-T system for tracking of intra-portal vein transplantation nanometer magnetically labeled BMSCs in rat liver. Methods: BMSCs were isolated from 5 SD rats bone marrow with the density gradient centrifugation method. Then BMSCs were labeled with nanometer superpara-magnetic iron oxide and transfection agent. Cell labeling efficiency was assessed with determination of the percentage of Peris Prussian blue stain. Then BMSCs transplanted into normal rats' livers via portal vein. The receipts were divided into 5 groups ,including sham control,2 h ,3 d,7 d and 2 w after transplantation. Follow-up serial T1WI,T2WI and T2*-weighted gradient- echo MR imaging were performed at 1.5 T MRI system. MR imaging findings were compared with histology. Results: Cell labeling efficiency was more than 95% by Perls Prussian blue stain. After transplantation of labeled BMSCs via portal vein, liver's had diffuse granular signal intensity appearance in T2* WI MRI. Cells were detected for up to 2 w in receipts' liver's. At histologic analysis, signal intensity loss correlated with iron-loaded cells. Conclusion: MR imaging could aid in monitoring of magnetically labeled BMSCs administered via portal vein in vivo. (authors)

  3. SCF increases in utero-labeled stem cells migration and improves wound healing.

    Science.gov (United States)

    Zgheib, Carlos; Xu, Junwang; Mallette, Andrew C; Caskey, Robert C; Zhang, Liping; Hu, Junyi; Liechty, Kenneth W

    2015-01-01

    Diabetic skin wounds lack the ability to heal properly and constitute a major and significant complication of diabetes. Nontraumatic lower extremity amputations are the number one complication of diabetic skin wounds. The complexity of their pathophysiology requires an intervention at many levels to enhance healing and wound closure. Stem cells are a promising treatment for diabetic skin wounds as they have the ability to correct abnormal healing. Stem cell factor (SCF), a chemokine expressed in the skin, can induce stem cells migration, however the role of SCF in diabetic skin wound healing is still unknown. We hypothesize that SCF would correct the impairment and promote the healing of diabetic skin wounds. Our results show that SCF improved wound closure in diabetic mice and increased HIF-1α and vascular endothelial growth factor (VEGF) expression levels in these wounds. SCF treatment also enhanced the migration of red fluorescent protein (RFP)-labeled skin stem cells via in utero intra-amniotic injection of lenti-RFP at E8. Interestingly these RFP+ cells are present in the epidermis, stain negative for K15, and appear to be distinct from the already known hair follicle stem cells. These results demonstrate that SCF improves diabetic wound healing in part by increasing the recruitment of a unique stem cell population present in the skin.

  4. Rapid and label-free separation of Burkitt's lymphoma cells from red blood cells by optically-induced electrokinetics.

    Directory of Open Access Journals (Sweden)

    Wenfeng Liang

    Full Text Available Early stage detection of lymphoma cells is invaluable for providing reliable prognosis to patients. However, the purity of lymphoma cells in extracted samples from human patients' marrow is typically low. To address this issue, we report here our work on using optically-induced dielectrophoresis (ODEP force to rapidly purify Raji cells' (a type of Burkitt's lymphoma cell sample from red blood cells (RBCs with a label-free process. This method utilizes dynamically moving virtual electrodes to induce negative ODEP force of varying magnitudes on the Raji cells and RBCs in an optically-induced electrokinetics (OEK chip. Polarization models for the two types of cells that reflect their discriminate electrical properties were established. Then, the cells' differential velocities caused by a specific ODEP force field were obtained by a finite element simulation model, thereby established the theoretical basis that the two types of cells could be separated using an ODEP force field. To ensure that the ODEP force dominated the separation process, a comparison of the ODEP force with other significant electrokinetics forces was conducted using numerical results. Furthermore, the performance of the ODEP-based approach for separating Raji cells from RBCs was experimentally investigated. The results showed that these two types of cells, with different concentration ratios, could be separated rapidly using externally-applied electrical field at a driven frequency of 50 kHz at 20 Vpp. In addition, we have found that in order to facilitate ODEP-based cell separation, Raji cells' adhesion to the OEK chip's substrate should be minimized. This paper also presents our experimental results of finding the appropriate bovine serum albumin concentration in an isotonic solution to reduce cell adhesion, while maintaining suitable medium conductivity for electrokinetics-based cell separation. In short, we have demonstrated that OEK technology could be a promising tool for

  5. Label-free detection of circulating melanoma cells by in vivo photoacoustic flow cytometry

    Science.gov (United States)

    Wang, Xiaoling; Yang, Ping; Liu, Rongrong; Niu, Zhenyu; Suo, Yuanzhen; He, Hao; Gao, Wenyuan; Tang, Shuo; Wei, Xunbin

    2016-03-01

    Melanoma is a malignant tumor of melanocytes. Melanoma cells have high light absorption due to melanin highly contained in melanoma cells. This property is employed for the detection of circulating melanoma cell by in vivo photoacoustic flow cytometry (PAFC), which is based on photoacoustic effect. Compared to in vivo flow cytometry based on fluorescence, PAFC can employ high melanin content of melanoma cells as endogenous biomarkers to detect circulating melanoma cells in vivo. We have developed in vitro experiments to prove the ability of PAFC system of detecting photoacoustic signals from melanoma cells. For in vivo experiments, we have constructed a model of melanoma tumor bearing mice by inoculating highly metastatic murine melanoma cancer cells, B16F10 with subcutaneous injection. PA signals are detected in the blood vessels of mouse ears in vivo. The raw signal detected from target cells often contains some noise caused by electronic devices, such as background noise and thermal noise. We choose the Wavelet denoising method to effectively distinguish the target signal from background noise. Processing in time domain and frequency domain would be combined to analyze the signal after denoising. This algorithm contains time domain filter and frequency transformation. The frequency spectrum image of the signal contains distinctive features that can be used to analyze the property of target cells or particles. The processing methods have a great potential for analyzing signals accurately and rapidly. By counting circulating melanoma cells termly, we obtain the number variation of circulating melanoma cells as melanoma metastasized. Those results show that PAFC is a noninvasive and label-free method to detect melanoma metastases in blood or lymph circulation.

  6. Correlative scanning electron and confocal microscopy imaging of labeled cells coated by indium-tin-oxide.

    Science.gov (United States)

    Rodighiero, Simona; Torre, Bruno; Sogne, Elisa; Ruffilli, Roberta; Cagnoli, Cinzia; Francolini, Maura; Di Fabrizio, Enzo; Falqui, Andrea

    2015-06-01

    Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field-of-view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold-labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium-tin-oxide was deposited by ion-sputtering on gold-decorated HeLa cells and neurons. Indium-tin-oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold-conjugated markers. PMID:25810353

  7. Correlative scanning electron and confocal microscopy imaging of labeled cells coated by indium-tin-oxide

    KAUST Repository

    Rodighiero, Simona

    2015-03-22

    Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field-of-view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold-labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium-tin-oxide was deposited by ion-sputtering on gold-decorated HeLa cells and neurons. Indium-tin-oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold-conjugated markers. © 2015 Wiley Periodicals, Inc.

  8. Direct in vivo cell lineage analysis in the retrorsine and 2AAF models of liver injury after genetic labeling in adult and newborn rats.

    Directory of Open Access Journals (Sweden)

    Virginie Pichard

    Full Text Available BACKGROUNDS AND AIMS: When hepatocyte proliferation is impaired, liver regeneration proceeds from the division of non parenchymal hepatocyte progenitors. Oval cells and Small Hepatocyte-like Progenitor Cells (SHPCs represent the two most studied examples of such epithelial cells with putative stem cell capacity. In the present study we wished to compare the origin of SHPCs proliferating after retrorsine administration to the one of oval cells observed after 2-Acetyl-Amino fluorene (2-AAF treatment. METHODOLOGY/PRINCIPAL FINDINGS: We used retroviral-mediated nlslacZ genetic labeling of dividing cells to study the fate of cells in the liver. Labeling was performed either in adult rats before treatment or in newborn animals. Labeled cells were identified and characterised by immunohistochemistry. In adult-labeled animals, labeling was restricted to mature hepatocytes. Retrorsine treatment did not modify the overall number of labeled cells in the liver whereas after 2-AAF administration unlabeled oval cells were recorded and the total number of labeled cells decreased significantly. When labeling was performed in newborn rats, results after retrorsine administration were identical to those obtained in adult-labeled rats. In contrast, in the 2-AAF regimen numerous labeled oval cells were present and were able to generate new labeled hepatocytes. Furthermore, we also observed labeled biliary tracts in 2-AAF treated rats. CONCLUSIONS: Our results strongly suggest that SHPCs are derived from hepatocytes and we confirm that SHPCs and oval cells do not share the same origin. We also show that hepatic progenitors are labeled in newborn rats suggesting future directions for in vivo lineage studies.

  9. MRI tracking of SPIO labelled stem cells in a true regenerative environment, the regenerating limb of the axolotl

    DEFF Research Database (Denmark)

    Lauridsen, Henrik; Foldager, Casper Bindzus; Hagensen, Mette;

    are generally restricted by their limited regenerative potential. Conversely, excellent animal models for regenerative studies exist in lower vertebrates such as the urodele amphibians (salamanders and newts), exemplified in the iconic Mexican axolotl (Ambystoma mexicanum) capable of regenerating whole limbs...... effect on blastema cell viability in vitro. MRI revealed that labelled tissue was clearly visibly in vivo 49 days after amputation and a significant decline in signal intensity of labelled limbs versus sham-operated limbs was evident throughout the complete regeneration period of 84 days. SPIO labelling...

  10. Enhancement of Raman light scattering in dye-labeled cell membrane on metal-containing conducting polymer film

    Science.gov (United States)

    Grushevskaya, H. V.; Krylova, N. G.; Lipnevich, I. V.; Orekhovskaja, T. I.; Egorova, V. P.; Shulitski, B. G.

    2016-03-01

    An enhanced Raman spectroscopy method based on a plasmon resonance in ultrathin metal-containing LB-film deposited on nanoporous anodic alumina supports has been proposed. This material has been utilized to enhance Raman scattering of light in fluorescent-labeled subcellular membrane structures. It has been shown that the plasmon resonance between vibrational modes of the organometallic complexes monolayers and dye-labeled subcellular structures happens. It makes possible to detect interactions between living cell monolayers and an extracellular matrix.

  11. Dynamic MRI of ferumoxide-labeled bone mesenchmal stem cells after transplantation in infarcted myocardium

    International Nuclear Information System (INIS)

    Objective: To investigate the ability of magnetic resonance imaging (MRI) in tracking magnetically labeled mesenchymal stem cells (MR-MSCs) in a swine myocardial infarction (MI) model. Methods: Adult Chinese mini-pigs (n=6) were subjected to open-chest experimental MI operation. Their autogeneic bone marrow-derived mesenchymal stem cells (MSCs) was cultured and doubly labeled with ferumoxides and DAPI. On the 14 th day after MSCs transplantation, the size and location of the myocardial infarction were assessed by using delayed-enhancement MRI (DE-MRI). Then the labeled MSCs were injected intramyocardially into peri-infarct zone and normal myocardium. At 24 hrs and 3 weeks after injection, the contrast and the volume of the MR-MSCs hypointense lesion from the MR images were acquired, and the contrast was determined using the difference in signal intensity between the hypointense and normal myocardium divided by signal intensity of the normal region. After humane euthanasia, the heart was excised and histology corresponding to MRI slices that demonstrated MR-MSCs lesions was performed. Repeated-measures ANOVA and a paired t test were used for comparison of the contrast and the volume of the MR-MSCs hypointense lesion at different time points. Comparisons between independent groups were performed with the standard Student t test. Results: The labeling efficiency of ferumoxides and DAPI was 100%. On the 14 th day after the MI operation, the average percentage of infracted myocardial area was (33.6±8.9)%. Twenty- four hours after MSCs transplantation, MSCs injection sites appeared as ovoid hypointensive lesions with sharp border on T2* images. At 24 h after injection, the signal contrast [(67.00±5.48)% vs (61.92±7.76)%,t=1.65, P=0.1158] and the size [(0.56±0.24) cm2 vs (0.52±0.25) cm2, t=0.39, P=0.7044] of the lesions showed no statistical difference between the peri-infarct zone and the normal myocardium. At 3 weeks after injection, the signal contrast decreased

  12. Highly Luminescent Heterostructured Copper-Doped Zinc Sulfide Nanocrystals for Application in Cancer Cell Labeling.

    Science.gov (United States)

    Ang, Huixiang; Bosman, Michel; Thamankar, Ramesh; Zulkifli, Muhammad Faizal B; Yen, Swee Kuan; Hariharan, Anushya; Sudhaharan, Thankiah; Selvan, Subramanian Tamil

    2016-08-18

    The structural characteristics of the seed-mediated synthesis of heterostructured CuS-ZnS nanocrystals (NCs) and Cu-doped ZnS (ZnS:Cu) NCs synthesized by two different protocols are compared and analyzed. At high Cu dopant concentrations, segregated subclusters of ZnS and CuS are observed. The photoluminescence quantum yield of ZnS:Cu NCs is about 50-80 %; a value much higher than that of ZnS NCs (6 %). Finally, these NCs are coated with a thin silica shell by using (3-mercaptopropyl)triethoxysilane in a reverse microemulsion to make them water soluble. Cytotoxicity experiments show that these silica-coated NCs have greatly reduced toxicity on both cancerous HeLa and noncancerous Chinese hamster ovary cells. The labeling of cancerous HeLa cells is also demonstrated. PMID:27146419

  13. Isoflavones in food supplements: chemical profile, label accordance and permeability study in Caco-2 cells.

    Science.gov (United States)

    Almeida, I M C; Rodrigues, F; Sarmento, B; Alves, R C; Oliveira, M B P P

    2015-03-01

    Consumers nowadays are playing an active role in their health-care. A special case is the increasing number of women, who are reluctant to use exogenous hormone therapy for the treatment of menopausal symptoms and are looking for complementary therapies. However, food supplements are not clearly regulated in Europe. The EFSA has only recently begun to address the issues of botanical safety and purity regulation, leading to a variability of content, standardization, dosage, and purity of available products. In this study, isoflavones (puerarin, daidzin, genistin, daidzein, glycitein, genistein, formononetin, prunetin, and biochanin A) from food supplements (n = 15) for menopausal symptoms relief are evaluated and compared with the labelled information. Only four supplements complied with the recommendations made by the EC on the tolerable thresholds. The intestinal bioavailability of these compounds was investigated using Caco-2 cells. The apparent permeability coefficients of the selected isoflavonoids across the Caco-2 cells were affected by the isoflavone concentration and product matrix. PMID:25653232

  14. Functionalized bismuth ferrite harmonic nanoparticles for cancer cells labeling and imaging

    Energy Technology Data Exchange (ETDEWEB)

    Passemard, Solène; Staedler, Davide; Sonego, Giona [Ecole Polytechnique Fédérale de Lausanne, Institute of Chemical Sciences and Engineering (Switzerland); Magouroux, Thibaud [Université de Genève, GAP-Biophotonics (Switzerland); Schneiter, Guillaume Stéphane [Ecole Polytechnique Fédérale de Lausanne, Institute of Chemical Sciences and Engineering (Switzerland); Juillerat-Jeanneret, Lucienne [University Institute of Pathology, CHUV-UNIL (Switzerland); Bonacina, Luigi [Université de Genève, GAP-Biophotonics (Switzerland); Gerber-Lemaire, Sandrine, E-mail: Sandrine.Gerber@epfl.ch [Ecole Polytechnique Fédérale de Lausanne, Institute of Chemical Sciences and Engineering (Switzerland)

    2015-10-15

    Bismuth ferrite (BFO) harmonic nanoparticles (NPs) display high nonlinear optical efficiency and excellent biocompatibility profile which make them attractive for the development of diagnostic applications as contrast agents. In this study, we present a general method for the functionalization of this material with chemical ligands targeting cancer molecular biomarkers. In particular, a conjugation protocol based on click reaction between alkynyl-containing targeting ligands and poly(ethylene glycol)-coated BFO NPs (67.7 nm) displaying surface reactive azido groups was developed. Copper-free click reaction allowed fast and efficient conjugation of a covalent inhibitor of prolyl-specific endopeptidases to coated BFO NPs. The ability of these functionalized nanomaterials (134.2 nm) to act as imaging probes for cancer cells was demonstrated by the selective labeling of human lung cancer cells.

  15. Labeling of human hepatocellular carcinoma cells by hexamethylene diamine modified fluorescent carbon dots

    Science.gov (United States)

    Dong, Wei; Dong, Yan; Wang, Ying; Zhou, Shiqi; Ge, Xin; Sui, Lili; Wang, Jingwen

    2013-12-01

    Fluorescent carbon dots (CDs) were synthesized by a solvothermal method with glucose as carbon source and surface-modified with 1,6-hexamethylene diamine. In this hybrid CDs, the modification played important role for improving the fluorescent performance by introducing nitrogenous compound to passivate CD's surface, making the CDs emit strong fluorescence. The as-prepared CDs were linked with mouse anti-human Alpha fetoprotein (AFP) antibody and goat anti-mouse immunoglobulin (IgG) to directly and indirectly label fixed human hepatocellular carcinoma cells, respectively. The cytotoxicity of these CDs were also tested using the human hepatocellular carcinoma cells. No apparent cytotoxicity was observed, which suggested the potential application of the as-prepared CDs in bioimaging.

  16. In vivo cell tracking imaging of hexadecyl-4-[{sup 123,} {sup 124}I]iodobenzoate labeled adipose derived stem cells (ADSCs) in rat heart

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Min Hwan; Lee, Yong Jin; Lee, Kyo Chul [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2011-10-15

    Monitoring of transplanted stem cells for cardiac repair is important part in regenerative medicine. Direct cell labeling techniques using [{sup 18}F]FDG, [{sup 64}Cu]PTSM and [{sup 99m}Tc]-HMPAO have been developed for in vivo imaging. Especially, {sup 18}F-labeled derivates have been widely used for direct labeling agent. But the {sup 18}F has short half life (T{sub 1/2}={approx}2 h), thus this imaging agent has limitation of in vivo imaging. We used {sup 123}I or {sup 124}I which has relative long half life, to track the transplanted stem cells for a long-term imaging. This study is aimed to track the transplanted adipose derived stem cells (ADSCs) in rat heart using hexadecyl-4-[{sup 123,} {sup 124}I]iodobenzoate ([{sup 123,} {sup 124}I]HIB) mediated direct labeling method in vivo

  17. A Breast Cell Atlas: Organelle analysis of the MDA-MB-231 cell line by density-gradient fractionation using isotopic marking and label-free analysis

    Directory of Open Access Journals (Sweden)

    Marianne Sandin

    2015-09-01

    Full Text Available Protein translocation between organelles in the cell is an important process that regulates many cellular functions. However, organelles can rarely be isolated to purity so several methods have been developed to analyse the fractions obtained by density gradient centrifugation. We present an analysis of the distribution of proteins amongst organelles in the human breast cell line, MDA-MB-231 using two approaches: an isotopic labelling and a label-free approach.

  18. Sickle Cell Research: Yesterday, Today, and Tomorrow

    Science.gov (United States)

    ... disease? Are there any restrictions on diet or exercise? Read More "Sickle Cell Disease" Articles "Be Sickle Smart!" / Sickle Cell Research: Yesterday, Today, and Tomorrow / Sickle Cell Disease: Symptoms, ...

  19. Integrating Cell Phone Imaging with Magnetic Levitation (i-LEV) for Label-Free Blood Analysis at the Point-of-Living.

    Science.gov (United States)

    Baday, Murat; Calamak, Semih; Durmus, Naside Gozde; Davis, Ronald W; Steinmetz, Lars M; Demirci, Utkan

    2016-03-01

    There is an emerging need for portable, robust, inexpensive, and easy-to-use disease diagnosis and prognosis monitoring platforms to share health information at the point-of-living, including clinical and home settings. Recent advances in digital health technologies have improved early diagnosis, drug treatment, and personalized medicine. Smartphones with high-resolution cameras and high data processing power enable intriguing biomedical applications when integrated with diagnostic devices. Further, these devices have immense potential to contribute to public health in resource-limited settings where there is a particular need for portable, rapid, label-free, easy-to-use, and affordable biomedical devices to diagnose and continuously monitor patients for precision medicine, especially those suffering from rare diseases, such as sickle cell anemia, thalassemia, and chronic fatigue syndrome. Here, a magnetic levitation-based diagnosis system is presented in which different cell types (i.e., white and red blood cells) are levitated in a magnetic gradient and separated due to their unique densities. Moreover, an easy-to-use, smartphone incorporated levitation system for cell analysis is introduced. Using our portable imaging magnetic levitation (i-LEV) system, it is shown that white and red blood cells can be identified and cell numbers can be quantified without using any labels. In addition, cells levitated in i-LEV can be distinguished at single-cell resolution, potentially enabling diagnosis and monitoring, as well as clinical and research applications. PMID:26523938

  20. Magentic Cell labeling of primary and stem cell-derived pig hepatocytes for MRI-based cell tracking of heptocytes transplantation

    Science.gov (United States)

    Pig hepatocytes are an important investigational tool for optimizing hepatocyte transplantation schemes in both allogeneic and xenogeneic transplant scenarios. MRI can be used to serially monitor the transplanted cells, but only if the hepatocytes can be labeled with a magnetic particle. In this wo...

  1. Lamotrigine increases the number of BrdU-labeled cells in the rat hippocampus

    DEFF Research Database (Denmark)

    Kondziella, Daniel; Strandberg, Joakim; Lindquist, Catarina;

    2011-01-01

    Antidepressant medication and electroconvulsive therapy stabilize mood symptoms and increase hippocampal neurogenesis. We examined whether lamotrigine, suggested to give rise to mood-stabilizing and antidepressant effects in addition to its antiepileptic properties, also increases the number of n...... in the granule cell layer of the dentate gyrus showed an increased number of newborn cells in rats receiving lamotrigine (42.6 ± 3.5 cells/slice) compared with valproate (31.6 ± 2.8) and controls (32.2 ± 3.1; P......Antidepressant medication and electroconvulsive therapy stabilize mood symptoms and increase hippocampal neurogenesis. We examined whether lamotrigine, suggested to give rise to mood-stabilizing and antidepressant effects in addition to its antiepileptic properties, also increases the number...... of newborn cells in rat hippocampus. Rats (on day P21) received lamotrigine, valproate, or saline intraperitoneally once daily for 7 days. All animals received four intraperitoneal injections of bromodeoxyuridine (BrdU) on day P28 and were sacrificed the next day. Quantification of BrdU-labeled cells...

  2. [Establishment and identification of the near-infrared fluorescence labeled exosomes in breast cancer cell lines].

    Science.gov (United States)

    Li, Taiming; Lan, Wenjun; Huang, Can; Zhang, Chun; Liu, Xiaomei

    2016-05-01

    Exosomes, a population of extracellular membrane vesicles of 30-100 nm in diameter, play important roles in cell biological functions, intercellular signal transduction and especially in cancer diagnosis and therapy. To better apply exosomes in mechanistic study of breast cancer signal transduction, we constructed recombinant eukaryotic expression vector expressing the near-infrared fluorescence protein and CD63 fusion protein through cloning iRFP682 gene and exosomal marker protein CD63 gene into plasmid containing the ITR of AAV. The constructed plasmids were co-transfected with helper plasmid in AAV-293 cell lines and were packaged into rAAV. After titer measurement, the recombinant plasmids were transfected into breast cancer cell lines. The cell lines that stably expressing near-infrared fluorescence protein were selected by fluorescence. Through isolation, purification and identification, we finally obtained a new biomarker: iRFP682 labeled exosomes secreted by breast cancer cell lines, which could be used in further studies of the distribution and signal transduction of exosomes in breast cancer microenvironment.

  3. Label-free identification of white blood cell using optical diffraction tomography (Conference Presentation)

    Science.gov (United States)

    Yoon, Jonghee; Kim, Kyoohyun; Kim, Min-hyeok; Kang, Suk-Jo; Park, YongKeun

    2016-03-01

    White blood cells (WBC) have crucial roles in immune systems which defend the host against from disease conditions and harmful invaders. Various WBC subsets have been characterized and reported to be involved in many pathophysiologic conditions. It is crucial to isolate a specific WBC subset to study its pathophysiological roles in diseases. Identification methods for a specific WBC population are rely on invasive approaches, including Wright-Gimesa staining for observing cellular morphologies and fluorescence staining for specific protein markers. While these methods enable precise classification of WBC populations, they could disturb cellular viability or functions. In order to classify WBC populations in a non-invasive manner, we exploited optical diffraction tomography (ODT). ODT is a three-dimensional (3-D) quantitative phase imaging technique that measures 3-D refractive index (RI) distributions of individual WBCs. To test feasibility of label-free classification of WBC populations using ODT, we measured four subtypes of WBCs, including B cell, CD4 T cell, CD8 T cell, and natural killer (NK) cell. From measured 3-D RI tomograms of WBCs, we obtain quantitative structural and biochemical information and classify each WBC population using a machine learning algorithm.

  4. Synthesis and evaluation of fluorine-18 labeled glyburide analogs as {beta}-cell imaging agents

    Energy Technology Data Exchange (ETDEWEB)

    Schmitz, A.; Shiue, C.-Y. E-mail: Shiue@rad.upenn.edu; Feng, Q.; Shiue, G.G.; Deng, S.; Pourdehnad, M.T.; Schirrmacher, R.; Vatamaniuk, M.; Doliba, N.; Matschinsky, F.; Wolf, B.; Roesch, F.; Naji, A.; Alavi, A.A

    2004-05-01

    Glyburide is a prescribed hypoglycemic drug for the treatment of type 2 diabetic patients. We have synthesized two of its analogs, namely N-{l_brace}4-[{beta}-(2-(2'-fluoroethoxy)-5-chlorobenzenecarboxamido)ethyl] benzenesulfonyl{r_brace}-N'-cyclohexylurea (2-fluoroethoxyglyburide, 8b) and N-{l_brace}4-[{beta}-(2-(2'-fluoroethoxy)-5-iodobenzenecarboxamido)ethyl]benzenesulfonyl {r_brace}-N'-cyclohexylurea (2-fluoroethoxy-5-deschloro-5-iodoglyburide, 8a), and their fluorine-18 labeled analogs as {beta}-cell imaging agents. Both F-18 labeled compound 8a and compound 8b were synthesized by alkylation of the corresponding multistep synthesized hydroxy precursor 4a and 4b with 2-[{sup 18}F]fluoroethyl tosylate in DMSO at 120 degree sign C for 20 minutes followed by HPLC purification in an overall radiochemical yield of 5-10% with a synthesis time of 100 minutes from EOB. The octanol/water partition coefficients of compounds 8a and 8b were 141.21 {+-} 27.77 (n = 8) and 124.33 {+-} 21.61 (n = 8), respectively. Insulin secretion experiments of compounds 8a and 8b on rat islets showed that both compounds have a similar stimulating effect on insulin secretion as that of glyburide. In vitro binding studies showed that {approx}2% of compounds 8a and 8b bound to {beta}TC3 and Min6 cells and that the binding was saturable. Preliminary biodistribution studies in mice showed that the uptake of both compounds 8a and 8b in liver and small intestine were high, whereas the uptake in other organs studied including pancreas were low. Additionally, the uptake of compound 8b in vivo was nonsaturable. These results tend to suggest that compounds 8a and 8b may not be the ideal {beta}-cell imaging agents.

  5. Selective cell-surface labeling of the molecular motor protein prestin

    Energy Technology Data Exchange (ETDEWEB)

    McGuire, Ryan M. [Department of Bioengineering, Rice University, Houston, TX 77251 (United States); Silberg, Jonathan J., E-mail: joff@rice.edu [Department of Bioengineering, Rice University, Houston, TX 77251 (United States); Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251 (United States); Pereira, Fred A. [Department of Bioengineering, Rice University, Houston, TX 77251 (United States); Huffington Center on Aging, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States); Raphael, Robert M., E-mail: rraphael@rice.edu [Department of Bioengineering, Rice University, Houston, TX 77251 (United States)

    2011-06-24

    Highlights: {yields} Trafficking to the plasma membrane is required for prestin function. {yields} Biotin acceptor peptide (BAP) was fused to prestin through a transmembrane domain. {yields} BAP-prestin can be metabolically labeled with biotin in HEK293 cells. {yields} Biotin-BAP-prestin allows for selective imaging of fully trafficked prestin. {yields} The biotin-BAP-prestin displays voltage-sensitive activity. -- Abstract: Prestin, a multipass transmembrane protein whose N- and C-termini are localized to the cytoplasm, must be trafficked to the plasma membrane to fulfill its cellular function as a molecular motor. One challenge in studying prestin sequence-function relationships within living cells is separating the effects of amino acid substitutions on prestin trafficking, plasma membrane localization and function. To develop an approach for directly assessing prestin levels at the plasma membrane, we have investigated whether fusion of prestin to a single pass transmembrane protein results in a functional fusion protein with a surface-exposed N-terminal tag that can be detected in living cells. We find that fusion of the biotin-acceptor peptide (BAP) and transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the N-terminus of prestin-GFP yields a membrane protein that can be metabolically-labeled with biotin, trafficked to the plasma membrane, and selectively detected at the plasma membrane using fluorescently-tagged streptavidin. Furthermore, we show that the addition of a surface detectable tag and a single-pass transmembrane domain to prestin does not disrupt its voltage-sensitive activity.

  6. Synthesis of carbon nanohorns/chitosan/quantum dots nanocomposite and its applications in cells labeling and in vivo imaging

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jing; He, Zhe [Chemistry Department, Northeastern University, Shenyang 110819 (China); Guo, Changrun [College of Life Sciences, Jilin University, Changchun 130023 (China); Wang, Liping, E-mail: wanglp@jlu.edu.cn [College of Life Sciences, Jilin University, Changchun 130023 (China); Xu, Shukun, E-mail: xushukun46@126.com [Chemistry Department, Northeastern University, Shenyang 110819 (China)

    2014-01-15

    Due to the unique optical and chemical features of quantum dots and the special structural advantages of carbon nanohorns, it is highly desirable to synthesize nanohorns/quantum dots nanocompsite which can be applied in cell labeling and in vivo imaging. Here, we report a new method which uses chitosan as connector to synthesize nanohorns/chitosan/quantum dots fluorescent nanocomposite. Further more, the synthesized nanocomposite demonstrated strong red fluorescence and had been successfully used in Hela cells labeling and in vivo imaging of Caenorhabditis elegans (C. elegans). -- Highlights: Carbon nanohorn/chitosan/QDs nanocomposite was prepared by covalent linkage The nanocomposite was successfully used in the labeling of HeLa cells The nanocomposite was used for in vivo imaging with C. elegans as animal mode.

  7. Fluorescently labeled adrenomedullin allows real-time monitoring of adrenomedullin receptor trafficking in living cells.

    Science.gov (United States)

    Schönauer, Ria; Kaiser, Anette; Holze, Cathleen; Babilon, Stefanie; Köbberling, Johannes; Riedl, Bernd; Beck-Sickinger, Annette G

    2015-12-01

    The human adrenomedullin (ADM) is a 52 amino acid peptide hormone belonging to the calcitonin family of peptides, which plays a major role in the development and regulation of cardiovascular and lymphatic systems. For potential use in clinical applications, we aimed to investigate the fate of the peptide ligand after binding and activation of the adrenomedullin receptor (AM1), a heterodimer consisting of the calcitonin receptor-like receptor (CLR), a G protein-coupled receptor, associated with the receptor activity-modifying protein 2 (RAMP2). Full length and N-terminally shortened ADM peptides were synthesized using Fmoc/tBu solid phase peptide synthesis and site-specifically labeled with the fluorophore carboxytetramethylrhodamine (Tam) either by amide bond formation or copper(I)-catalyzed azide alkyne cycloaddition. For the first time, Tam-labeled ligands allowed the observation of co-internalization of the whole ligand-receptor complex in living cells co-transfected with fluorescent fusion proteins of CLR and RAMP2. Application of a fluorescent probe to track lysosomal compartments revealed that ADM together with the CLR/RAMP2-complex is routed to the degradative pathway. Moreover, we found that the N-terminus of ADM is not a crucial component of the peptide sequence in terms of AM1 internalization behavior. PMID:26767744

  8. In vitro targeted magnetic delivery and tracking of superparamagnetic iron oxide particles labeled stem cells for articular cartilage defect repair.

    Science.gov (United States)

    Feng, Yong; Jin, Xuhong; Dai, Gang; Liu, Jun; Chen, Jiarong; Yang, Liu

    2011-04-01

    To assess a novel cell manipulation technique of tissue engineering with respect to its ability to augment superparamagnetic iron oxide particles (SPIO) labeled mesenchymal stem cells (MSCs) density at a localized cartilage defect site in an in vitro phantom by applying magnetic force. Meanwhile, non-invasive imaging techniques were use to track SPIO-labeled MSCs by magnetic resonance imaging (MRI). Human bone marrow MSCs were cultured and labeled with SPIO. Fresh degenerated human osteochondral fragments were obtained during total knee arthroplasty and a cartilage defect was created at the center. Then, the osteochondral fragments were attached to the sidewalls of culture flasks filled with phosphate-buffered saline (PBS) to mimic the human joint cavity. The SPIO-labeled MSCs were injected into the culture flasks in the presence of a 0.57 Tesla (T) magnetic force. Before and 90 min after cell targeting, the specimens underwent T2-weighted turbo spin-echo (SET2WI) sequence of 3.0 T MRI. MRI results were compared with histological findings. Macroscopic observation showed that SPIO-labeled MSCs were steered to the target region of cartilage defect. MRI revealed significant changes in signal intensity (P<0.01). HE staining exibited that a great number of MSCs formed a three-dimensional (3D) cell "sheet" structure at the chondral defect site. It was concluded that 0.57 T magnetic force permits spatial delivery of magnetically labeled MSCs to the target region in vitro. High-field MRI can serve as an very sensitive non-invasive technique for the visualization of SPIO-labeled MSCs.

  9. Efficient nano iron particle-labeling and noninvasive MR imaging of mouse bone marrow-derived endothelial progenitor cells

    Directory of Open Access Journals (Sweden)

    Zhen-Yu Jia

    2011-03-01

    Full Text Available Rong Chen*, Hui Yu*, Zhen-Yu Jia, Qun-Li Yao, Gao-Jun TengJiangsu Key Laboratory of Molecular Imaging and Functional Imaging, Department of Radiology, Zhongda Hospital, Medical School of Southeast University, Nanjing, People’s Republic of China *These authors have contributed equally to this workAbstract: In this study, we sought to label mouse bone marrow-derived endothelial progenitor cells (EPCs with Resovist® in vitro and to image them using 7.0 Tesla (T magnetic resonance imaging (MRI. Mouse bone marrow-derived EPCs were cultured in endothelial basal medium with endothelial growth supplement. They were then characterized by immunocytochemistry, flow cytometry, and fluorescence quantitative polymerase chain reaction. Their functions were evaluated by measuring their uptake of 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (Dil-Ac-LDL, binding of fluorine isothiocyanate (FITC-labeled Ulex europaeus agglutinin (UEA, and formation of capillary-like networks. EPCs were labeled with superparamagnetic iron oxide (SPIO and their proliferation was then assessed in a water-soluble tetrazolium (WST-8-based cell proliferation assay. Spin echo sequence (multislice, multiecho [MSME] and gradient echo sequence (2D-FLASH were used to detect differences in the numbers of labeled cells by 7.0 T MRI. The results showed that the cultured cells were of “cobblestone”-like shape and positive for CD133, CD34, CD31, von Willebrand factor, kinase domain receptor, and CD45, but negative for F4/80. They could take up Dil-Ac-LDL, bind FITC-UEA, and form capillary-like networks on Matrigel in vitro. Prussian-blue staining demonstrated that the cells were efficiently labeled with SPIO. The single-cell T2* effect was more obvious in the 2D-FLASH sequence than in the MSME sequence. Further, there were almost no adverse effects on cell vitality and proliferation. In conclusion, mouse bone marrow-derived EPCs can be

  10. Long-term MRI tracking of dual-labeled adipose-derived stem cells homing into mouse carotid artery injury

    Directory of Open Access Journals (Sweden)

    Qin JB

    2012-10-01

    Full Text Available Jin-Bao Qin,1,5,* Kang-An Li,2,* Xiang-Xiang Li,1,5 Qing-Song Xie,3 Jia-Ying Lin,4 Kai-Chuang Ye,1,5 Mi-Er Jiang,1,5 Gui-Xiang Zhang,2 Xin-Wu Lu1,51Department of Vascular Surgery, Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University, School of Medicine, 2Department of Radiology, Shanghai First People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 3Department of Neurosurgery, Cixi Municipal People's Hospital, Zhejiang Province, China; 4Clinic for Gynecology, Charite-Universitatsmedizin Berlin, Berlin, Germany; 5Vascular Center, Shanghai Jiao Tong University, Shanghai, China*These two authors contributed equally to this workBackground: Stem cell therapy has shown great promise for regenerative repair of injured or diseased tissues. Adipose-derived stem cells (ADSCs have become increasingly attractive candidates for cellular therapy. Magnetic resonance imaging has been proven to be effective in tracking magnetic-labeled cells and evaluating their clinical relevance after cell transplantation. This study investigated the feasibility of imaging green fluorescent protein-expressing ADSCs (GFP-ADSCs labeled with superparamagnetic iron oxide particles, and tracked them in vivo with noninvasive magnetic resonance imaging after cell transplantation in a model of mouse carotid artery injury.Methods: GFP-ADSCs were isolated from the adipose tissues of GFP mice and labeled with superparamagnetic iron oxide particles. Intracellular stability, proliferation, and viability of the labeled cells were evaluated in vitro. Next, the cells were transplanted into a mouse carotid artery injury model. Clinical 3 T magnetic resonance imaging was performed immediately before and 1, 3, 7, 14, 21, and 30 days after cell transplantation. Prussian blue staining and histological analysis were performed 7 and 30 days after transplantation.Results: GFP-ADSCs were found to be efficiently labeled with superparamagnetic iron oxide

  11. Tissue distribution of adoptively transferred adherent lymphokine-activated killer cells assessed by different cell labels

    DEFF Research Database (Denmark)

    Basse, P; Herberman, R B; Hokland, M;

    1992-01-01

    by alternative direct visual methods for identification of the injected cells, such as fluorescent dyes (rhodamine and H33342) or immunohistochemical staining of asialo-GM1-positive cells. The number of i.v. injected A-LAK cells found in the liver by all visual methods ranged from 1% to 5% of the injected dose...

  12. Divergent Label-free Cell Phenotypic Pharmacology of Ligands at the Overexpressed β2-Adrenergic Receptors

    Science.gov (United States)

    Ferrie, Ann M.; Sun, Haiyan; Zaytseva, Natalya; Fang, Ye

    2014-01-01

    We present subclone sensitive cell phenotypic pharmacology of ligands at the β2-adrenergic receptor (β2-AR) stably expressed in HEK-293 cells. The parental cell line was transfected with green fluorescent protein (GFP)-tagged β2-AR. Four stable subclones were established and used to profile a library of sixty-nine AR ligands. Dynamic mass redistribution (DMR) profiling resulted in a pharmacological activity map suggesting that HEK293 endogenously expresses functional Gi-coupled α2-AR and Gs-coupled β2-AR, and the label-free cell phenotypic activity of AR ligands are subclone dependent. Pathway deconvolution revealed that the DMR of epinephrine is originated mostly from the remodeling of actin microfilaments and adhesion complexes, to less extent from the microtubule networks and receptor trafficking, and certain agonists displayed different efficacy towards the cAMP-Epac pathway. We demonstrate that receptor signaling and ligand pharmacology is sensitive to the receptor expression level, and the organization of the receptor and its signaling circuitry.

  13. Rapid kinetic labeling of Arabidopsis cell suspension cultures: Implications for models of lipid export from plastids

    Science.gov (United States)

    T-87 suspension cell cultures are increasingly used in Arabidopsis research, but there are no reports describing their lipid composition or biosynthesis. To evaluate if T-87 cell cultures as a model system for analysis of lipid metabolism, including tests of gene candidate functions, we have deter...

  14. Use of chromium-50 as a label for red blood cells in studies with pregnant women and premature infants

    International Nuclear Information System (INIS)

    A technique is described in which non-radioactive chromium-50 is used as a label for red blood cells in patients for whom radioactive labels are not permissible. The chromium-50, adsorbed on to donor blood in vitro, is infused in the circulatory system and measured, following collection, using neutron activation analyses and a high resolution germanium (lithium) diode gamma-ray spectrometer. The application of this technique to the measurement of blood cell survival time in pregnant women suspected of having haemolytic anaemia and to the measurement of intracranial bleeding in premature infants is described. (author)

  15. Resonance Raman Probes for Organelle-Specific Labeling in Live Cells

    Science.gov (United States)

    Kuzmin, Andrey N.; Pliss, Artem; Lim, Chang-Keun; Heo, Jeongyun; Kim, Sehoon; Rzhevskii, Alexander; Gu, Bobo; Yong, Ken-Tye; Wen, Shangchun; Prasad, Paras N.

    2016-06-01

    Raman microspectroscopy provides for high-resolution non-invasive molecular analysis of biological samples and has a breakthrough potential for dissection of cellular molecular composition at a single organelle level. However, the potential of Raman microspectroscopy can be fully realized only when novel types of molecular probes distinguishable in the Raman spectroscopy modality are developed for labeling of specific cellular domains to guide spectrochemical spatial imaging. Here we report on the design of a next generation Raman probe, based on BlackBerry Quencher 650 compound, which provides unprecedentedly high signal intensity through the Resonance Raman (RR) enhancement mechanism. Remarkably, RR enhancement occurs with low-toxic red light, which is close to maximum transparency in the biological optical window. The utility of proposed RR probes was validated for targeting lysosomes in live cultured cells, which enabled identification and subsequent monitoring of dynamic changes in this organelle by Raman imaging.

  16. Spermatogonial stem cell renewal in the mouse as revealed by 3H-thymidine labeling and irradiation

    International Nuclear Information System (INIS)

    Procedures are described for sectioning and staining mouse testes, x-irradiation of mice, and classification of developing sperm cells. Tables are presented to show frequency of labeled cells following exposure to various doses of x radiation. A discussion is presented of types of spermatogonia surviving various radiation doses and times of incorporation of 3H-TdR in the cycle of the seminiferous epithelium. Models of stem cell renewal systems are described

  17. Magnetic resonance imaging tracking of ferumoxytol-labeled human neural stem cells: studies leading to clinical use.

    Science.gov (United States)

    Gutova, Margarita; Frank, Joseph A; D'Apuzzo, Massimo; Khankaldyyan, Vazgen; Gilchrist, Megan M; Annala, Alexander J; Metz, Marianne Z; Abramyants, Yelena; Herrmann, Kelsey A; Ghoda, Lucy Y; Najbauer, Joseph; Brown, Christine E; Blanchard, M Suzette; Lesniak, Maciej S; Kim, Seung U; Barish, Michael E; Aboody, Karen S; Moats, Rex A

    2013-10-01

    Numerous stem cell-based therapies are currently under clinical investigation, including the use of neural stem cells (NSCs) as delivery vehicles to target therapeutic agents to invasive brain tumors. The ability to monitor the time course, migration, and distribution of stem cells following transplantation into patients would provide critical information for optimizing treatment regimens. No effective cell-tracking methodology has yet garnered clinical acceptance. A highly promising noninvasive method for monitoring NSCs and potentially other cell types in vivo involves preloading them with ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) to enable cell tracking using magnetic resonance imaging (MRI). We report here the preclinical studies that led to U.S. Food and Drug Administration approval for first-in-human investigational use of ferumoxytol to label NSCs prior to transplantation into brain tumor patients, followed by surveillance serial MRI. A combination of heparin, protamine sulfate, and ferumoxytol (HPF) was used to label the NSCs. HPF labeling did not affect cell viability, growth kinetics, or tumor tropism in vitro, and it enabled MRI visualization of NSC distribution within orthotopic glioma xenografts. MRI revealed dynamic in vivo NSC distribution at multiple time points following intracerebral or intravenous injection into glioma-bearing mice that correlated with histological analysis. Preclinical safety/toxicity studies of intracerebrally administered HPF-labeled NSCs in mice were also performed, and they showed no significant clinical or behavioral changes, no neuronal or systemic toxicities, and no abnormal accumulation of iron in the liver or spleen. These studies support the clinical use of ferumoxytol labeling of cells for post-transplant MRI visualization and tracking.

  18. C d 1a-labeled Langerhans Cells in Gingival Pyogenic Granuloma and Fibroma Lesions: An Immunohistochemical Study

    Directory of Open Access Journals (Sweden)

    Arpan K Shah

    2015-07-01

    Full Text Available Background: Role of Langerhans cells is well investigated in pathological conditions of oral tissues. No studies have been reported so far describing possible role of Langerhans cells in reactive gingival overgr owths. This study aims at evaluation of density of Langerhans cells in common localized gingival overgrowths. Materials and methods: Thirty-two specimens of localized gingival overgrowth s (19 fibromas and 13 pyogenic granulo - mas were subjected to immunohistochemistry with anti-CD1a antibody for demonstration of Langerhans cells. Langerhans cells in each specimen were counted as number of cells/high power field. The density of Langerhans cells thus obtained in study groups was compar ed. It was also compared with the same in control group. Results: Mean number of CD1a-labeled cells in control group was 2.24 ± 1.11 cells/HPF. The mean number of CD1a-labeled cells was 2.39 ± 1.80 cells/HPF in pyogenic granuloma group, while in irritation fibroma group the mean number was as 1.71 ± 1.96 cells/HPF. The difference in the density of Langerhans cells among two stud y groups was not significant. Conclusion: Langerhans cells were an integral component of epithelium in both types of reactive lesions. A wide range in density of Langerhans cells was observed in both groups.

  19. Thyroid cell lines in research on goitrogenesis.

    Science.gov (United States)

    Gerber, H; Peter, H J; Asmis, L; Studer, H

    1991-12-01

    Thyroid cell lines have contributed a lot to the understanding of goitrogenesis. The cell lines mostly used in thyroid research are briefly discussed, namely the rat thyroid cell lines FRTL and FRTL-5, the porcine thyroid cell lines PORTHOS and ARTHOS, The sheep thyroid cell lines OVNIS 5H and 6H, the cat thyroid cell lines PETCAT 1 to 4 and ROMCAT, and the human thyroid cell lines FTC-133 and HTh 74. Chinese hamster ovary (CHO) cells and COS-7 cells, stably transfected with TSH receptor cDNA and expressing a functional TSH receptor, are discussed as examples for non-thyroidal cells, transfected with thyroid genes. PMID:1726925

  20. Surfactant-free Gd3+-ion-containing carbon nanotube MRI contrast agents for stem cell labeling

    Science.gov (United States)

    Gizzatov, Ayrat; Hernández-Rivera, Mayra; Keshishian, Vazrik; Mackeyev, Yuri; Law, Justin J.; Guven, Adem; Sethi, Richa; Qu, Feifei; Muthupillai, Raja; Cabreira-Hansen, Maria Da Graça; Willerson, James T.; Perin, Emerson C.; Ma, Qing; Bryant, Robert G.; Wilson, Lon J.

    2015-07-01

    There is an ever increasing interest in developing new stem cell therapies. However, imaging and tracking stem cells in vivo after transplantation remains a serious challenge. In this work, we report new, functionalized and high-performance Gd3+-ion-containing ultra-short carbon nanotube (US-tube) MRI contrast agent (CA) materials which are highly-water-dispersible (ca. 35 mg ml-1) without the need of a surfactant. The new materials have extremely high T1-weighted relaxivities of 90 (mM s)-1 per Gd3+ ion at 1.5 T at room temperature and have been used to safely label porcine bone-marrow-derived mesenchymal stem cells for MR imaging. The labeled cells display excellent image contrast in phantom imaging experiments, and TEM images of the labeled cells, in general, reveal small clusters of the CA material located within the cytoplasm with 109 Gd3+ ions per cell.There is an ever increasing interest in developing new stem cell therapies. However, imaging and tracking stem cells in vivo after transplantation remains a serious challenge. In this work, we report new, functionalized and high-performance Gd3+-ion-containing ultra-short carbon nanotube (US-tube) MRI contrast agent (CA) materials which are highly-water-dispersible (ca. 35 mg ml-1) without the need of a surfactant. The new materials have extremely high T1-weighted relaxivities of 90 (mM s)-1 per Gd3+ ion at 1.5 T at room temperature and have been used to safely label porcine bone-marrow-derived mesenchymal stem cells for MR imaging. The labeled cells display excellent image contrast in phantom imaging experiments, and TEM images of the labeled cells, in general, reveal small clusters of the CA material located within the cytoplasm with 109 Gd3+ ions per cell. Electronic supplementary information (ESI) available: NMRD profiles, the Fourier transforms of the EXAFS data, EXAFS curve fitting data, cell viability data. See DOI: 10.1039/c5nr02078f

  1. Setting FIRES to Stem Cell Research

    Science.gov (United States)

    Miller, Roxanne Grietz

    2005-01-01

    The goal of this lesson is to present the basic scientific knowledge about stem cells, the promise of stem cell research to medicine, and the ethical considerations and arguments involved. One of the challenges of discussing stem cell research is that the field is constantly evolving and the most current information changes almost daily. Few…

  2. Iron labeling and pre-clinical MRI visualization of therapeutic human neural stem cells in a murine glioma model.

    Directory of Open Access Journals (Sweden)

    Mya S Thu

    Full Text Available BACKGROUND: Treatment strategies for the highly invasive brain tumor, glioblastoma multiforme, require that cells which have invaded into the surrounding brain be specifically targeted. The inherent tumor-tropism of neural stem cells (NSCs to primary and invasive tumor foci can be exploited to deliver therapeutics to invasive brain tumor cells in humans. Use of the strategy of converting prodrug to drug via therapeutic transgenes delivered by immortalized therapeutic NSC lines have shown efficacy in animal models. Thus therapeutic NSCs are being proposed for use in human brain tumor clinical trials. In the context of NSC-based therapies, MRI can be used both to non-invasively follow dynamic spatio-temporal patterns of the NSC tumor targeting allowing for the optimization of treatment strategies and to assess efficacy of the therapy. Iron-labeling of cells allows their presence to be visualized and tracked by MRI. Thus we aimed to iron-label therapeutic NSCs without affecting their cellular physiology using a method likely to gain United States Federal Drug Administration (FDA approval. METHODOLOGY: For human use, the characteristics of therapeutic Neural Stem Cells must be clearly defined with any pertubation to the cell including iron labeling requiring reanalysis of cellular physiology. Here, we studied the effect of iron-loading of the therapeutic NSCs, with ferumoxide-protamine sulfate complex (FE-Pro on viability, proliferation, migratory properties and transgene expression, when compared to non-labeled cells. FE-Pro labeled NSCs were imaged by MRI at tumor sites, after intracranial administration into the hemisphere contralateral to the tumor, in an orthotopic human glioma xenograft mouse model. CONCLUSION: FE-Pro labeled NSCs retain their proliferative status, tumor tropism, and maintain stem cell character, while allowing in vivo cellular MRI tracking at 7 Tesla, to monitor their real-time migration and distribution at brain tumor sites

  3. Affordable uniform isotope labeling with {sup 2}H, {sup 13}C and {sup 15}N in insect cells

    Energy Technology Data Exchange (ETDEWEB)

    Sitarska, Agnieszka; Skora, Lukasz; Klopp, Julia; Roest, Susan; Fernández, César; Shrestha, Binesh; Gossert, Alvar D., E-mail: alvar.gossert@novartis.com [Novartis Institutes for BioMedical Research (Switzerland)

    2015-06-15

    For a wide range of proteins of high interest, the major obstacle for NMR studies is the lack of an affordable eukaryotic expression system for isotope labeling. Here, a simple and affordable protocol is presented to produce uniform labeled proteins in the most prevalent eukaryotic expression system for structural biology, namely Spodoptera frugiperda insect cells. Incorporation levels of 80 % can be achieved for {sup 15}N and {sup 13}C with yields comparable to expression in full media. For {sup 2}H,{sup 15}N and {sup 2}H,{sup 13}C,{sup 15}N labeling, incorporation is only slightly lower with 75 and 73 %, respectively, and yields are typically twofold reduced. The media were optimized for isotope incorporation, reproducibility, simplicity and cost. High isotope incorporation levels for all labeling patterns are achieved by using labeled algal amino acid extracts and exploiting well-known biochemical pathways. The final formulation consists of just five commercially available components, at costs 12-fold lower than labeling media from vendors. The approach was applied to several cytosolic and secreted target proteins.

  4. Isolation and (111)In-Oxine Labeling of Murine NK Cells for Assessment of Cell Trafficking in Orthotopic Lung Tumor Model.

    Science.gov (United States)

    Malviya, Gaurav; Nayak, Tapan; Gerdes, Christian; Dierckx, Rudi A J O; Signore, Alberto; de Vries, Erik F J

    2016-04-01

    A noninvasive in vivo imaging method for NK cell trafficking is essential to gain further understanding of the pathogenesis of NK cell mediated immune response to the novel cancer treatment strategies, and to discover the homing sites and physiological distribution of NK cells. Although human NK cells can be labeled for in vivo imaging, little is known about the murine NK cell labeling and its application in animal models. This study describes the isolation and ex vivo radiolabeling of murine NK cells for the evaluation of cell trafficking in an orthotopic model of human lung cancer in mice. Scid-Tg(FCGR3A)Blt transgenic SCID mice were used to isolate NK cells from mouse splenocytes using the CD49b (DX5) MicroBeads positive selection method. The purity and viability of the isolated NK cells were confirmed by FACS analysis. Different labeling buffers and incubation times were evaluated to optimize (111)In-oxine labeling conditions. Functionality of the radiolabeled NK cell was assessed by (51)Cr-release assay. We evaluated physiological distribution of (111)In-oxine labeled murine NK cells in normal SCID mice and biodistribution in irradiated and nonirradiated SCID mice with orthotopic A549 human lung tumor lesions. Imaging findings were confirmed by histology. Results showed that incubation with 0.011 MBq of (111)In-oxine per million murine NK cells in PBS (pH 7.4) for 20 min is the best condition that provides optimum labeling efficiency without affecting cell viability and functionality. Physiological distribution in normal SCID mice demonstrated NK cells homing mainly in the spleen, while (111)In released from NK cells was excreted via kidneys into urine. Biodistribution studies demonstrated a higher lung uptake in orthotopic lung tumor-bearing mice than control mice. In irradiated mice, lung tumor uptake of radiolabeled murine NK cells decreased between 24 h and 72 h postinjection (p.i.), which was accompanied by tumor regression, while in nonirradiated mice

  5. Radioactively labelled DNA probes for crop improvement. Proceedings of a final research co-ordination meeting

    International Nuclear Information System (INIS)

    With the advent of DNA molecular marker technology in the 1980s plant breeding had a new and powerful tool with which to increase its efficacy. Such markers are abundant and directly reveal information about the genotype and therefore are more useful than simple phenotypic markers. In plant breeding applications, molecular markers reveal information about variability and genetic relationships, and enable genetic mapping, which greatly assists the breeder in selection of parents and progeny, as well as in management of breeding strategies. Furthermore, molecular markers linked to phenotypic traits permit very early selection of superior progenies from breeding populations, therefore significantly reducing the need for field testing and greatly increasing efficiency of plant breeding programmes. For this to occur the oligonucleotide probes for labelling genetic markers and/or the primers for polymerase chain reactions to amplify genetic markers needed to be also accessible to scientists in developing Member States. In addition, technical information, training and troubleshooting were needed to support the utilization of DNA markers. In the early 1990s there was a dramatic increase in requests for access to this technology. This co-ordinated research project (CRP) facilitated the transfer of molecular marker technology, in terms of both material and information, from advanced laboratories to assist breeding programmes in developing countries. Two other CRPs were conducted concurrently in order to assist developing Member States to utilise molecular markers - Application of DNA Based Marker Mutations for Improvement of Cereals and other Sexually Reproduced Crop Plants, and Use of Novel DNA Fingerprinting Techniques for the Detection and Characterisation of Genetic Variation in Vegetatively Propagated Crops (IAEA-TECDOC-1010 and IAEA-TECDOC-1047, respectively). The present CRP built upon the success of the former projects by ensuring the availability of probes

  6. Detection of homing-in of stem cells labeled with technetium-99m hexamethylpropyleneamine oxime in infarcted myocardium after intracoronary injection

    International Nuclear Information System (INIS)

    Bone marrow stem cells having myogenic potential are promising candidates for various cell-based therapies for myocardial disease. We present here images showing homing of technetium-99m (Tc-99m) hexamethylpropyleneamine oxime (HMPAO) labeled stem cells in the infarcted myocardium from a pilot study conducted to radio-label part of the stem cells in patients enrolled in a stem cell clinical trial for recent myocardial infarction

  7. Effect of DiD Carbocyanine Dye Labeling on Immunoregulatory Function and Differentiation of Mice Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Maryam Sadat Mohtasebi

    2014-01-01

    Full Text Available Mesenchymal stem cells (MSCs have been used to treat a variety of degenerative disorders. Labeling of MSCs with an appropriate tracer is vital to demonstrate the in vivo engraftment and differentiation of transplanted MSCs. DiD is a lipophilic fluorescent dye with near infrared emission spectra that makes it suitable for in vivo tracing. Therefore, in the present study the consequences of DiD labeling on induction of oxidative stress and apoptosis as well as inhibition of biological functions of mesenchymal stem cells (MSCs were investigated. DiD labeling did not provoke the production of ROS, induction of apoptosis, or inhibition of production of immunosuppressive factors (PGE2 and IL-10 by MSCs. In addition, there were no statistical differences between DiD-labeled and unlabeled MSCs in suppression of proliferation and cytokine production (IFN-γ and IL-17 by in vitro stimulated splenocytes or improvement of clinical score in EAE after in vivo administration. In addition, DiD labeling did not alter the differentiation capacity of MSCs. Taken together, DiD can be considered as a safe dye for in vivo tracking of MSCs.

  8. A Novel PET Imaging Using 64Cu-Labeled Monoclonal Antibody against Mesothelin Commonly Expressed on Cancer Cells

    Directory of Open Access Journals (Sweden)

    Kazuko Kobayashi

    2015-01-01

    Full Text Available Mesothelin (MSLN is a 40-kDa cell differentiation-associated glycoprotein appearing with carcinogenesis and is highly expressed in many human cancers, including the majority of pancreatic adenocarcinomas, ovarian cancers, and mesotheliomas, while its expression in normal tissue is limited to mesothelial cells lining the pleura, pericardium, and peritoneum. Clone 11-25 is a murine hybridoma secreting monoclonal antibody (mAb against human MSLN. In this study, we applied the 11-25 mAb to in vivo imaging to detect MSLN-expressing tumors. In in vitro and ex vivo immunochemical studies, we demonstrated specificity of 11-25 mAb to membranous MSLN expressed on several pancreatic cancer cells. We showed the accumulation of Alexa Fluor 750-labeled 11-25 mAb in MSLN-expressing tumor xenografts in athymic nude mice. Then, 11-25 mAb was labeled with 64Cu via a chelating agent DOTA and was used in both in vitro cell binding assay and in vivo positron emission tomography (PET imaging in the tumor-bearing mice. We confirmed that 64Cu-labeled 11-25 mAb highly accumulated in MSLN-expressing tumors as compared to MSLN-negative ones. The 64Cu-labeled 11-25 mAb is potentially useful as a PET probe capable of being used for wide range of tumors, rather than 18F-FDG that occasionally provides nonspecific accumulation into the inflammatory lesions.

  9. Detection of clonal T cell populations by high resolution PCR using fluorescently labelled nucleotides; evaluation using conventional LIS-SSCP

    OpenAIRE

    Wickham, C L; Lynas, C; Ellard, S

    2000-01-01

    Aims—To detect clonal T cell populations by high resolution polymerase chain reaction (PCR) using fluorescently labelled nucleotides and analysis on an ABI 377 DNA sequencer, and to evaluate this method using low ionic strength single strand conformation polymorphism (LIS-SSCP) analysis.

  10. Gallbladder Activity on 99mTc-Labeled Red Cell Scintigraphy Confirmed by SPECT/CT Imaging.

    Science.gov (United States)

    Wang, Ling; Jing, Hongli; Chen, Libo; Wang, Zhenghua; Li, Fang

    2016-09-01

    Tc-labeled red cell (Tc-RBC) scintigraphy is commonly used to detect gastrointestinal bleeding. Gallbladder visualization on Tc-RBC scintigraphy is not common. We present a case of gallbladder visualization on Tc-RBC scintigraphy confirmed by SPECT/CT imaging in a patient with chronic renal failure and anemia. PMID:27405034

  11. Location of tumour cells in colon tissue by Texas red labelled pentosan polysulphate, an inhibitor of a cell surface protease.

    Science.gov (United States)

    Anees, M

    1996-01-01

    Pentosan polysulphate (PPS), a highly negatively charged polysaccharide, is a significant inhibitor of an isoenzymic form of a cell surface protease referred to as guanidinobenzoatase GB, associated with colonic carcinoma tissues in frozen sections and free GB in solution, in a concentration-dependent manner. However PPS failed to recognise and bind to the isoenzymic form of GB associated with normal colon epithelial cell surfaces. Texas red labelled PPS (TR-PPS) binds to the tumour cell surfaces of colonic carcinoma and colonic polyps and these cells fluoresce red, whilst the normal colon cell surfaces failed to bind the TR-PPS, and hence lacked red fluorescence. Polysulphonated suramin also selectively recognised and inhibited the colonic carcinoma GB isoenzyme. The kinetic data indicated that this inhibition was not caused by a mere polyanionic effect, since highly sulphated heparin failed to show a significant inhibition of colonic carcinoma GB, however trypan blue did show 50% inhibition. Kinetic studies have also shown that PPS is a non-competitive, reversible inhibitor of colonic carcinoma GB, with an apparent Km 6.8 x 10(-7) M. Gel analysis has shown that PPS binds to another site, distinct from the active centre, and after binding PPS changed the conformation of GB. These studies suggest that TR-PPS is a potent inhibitor of colonic carcinoma GB, and can be used as a novel fluorescent probe for the location of tumour cells in frozen sections of human colon tissues. PSS could also have potential as a vehicle for the transport of cytotoxic compounds to carcinoma cells of the colon. PMID:8835946

  12. Label-Free Digital Quantification of Lipid Droplets in Single Cells by Stimulated Raman Microscopy on a Microfluidic Platform.

    Science.gov (United States)

    Cao, Chen; Zhou, Dong; Chen, Tao; Streets, Aaron M; Huang, Yanyi

    2016-05-01

    Quantitative characterization of a single-cell phenotype remains challenging. We combined a scalable microfluidic array of parallel cell culture chambers and stimulated Raman scattering (SRS) microscopy to quantitatively characterize the response of lipid droplet (LD) formation to free-fatty-acid stimuli with single-LD resolution at the single-cell level. By enabling the systematic live-cell imaging with SRS microscopy in a microfluidic device, we were able to quantify the morphology of over a thousand live cells in 10 different chemical environments and with 8 replicates for each culture condition, in a single experiment, and without relying on fluorescent labeling. We developed an image processing pipeline for cell segmentation and LD morphology quantification using dual-channel SRS images. This allows us to construct distributions of the morphological parameters of LDs in the cellular population and expose the vast phenotypic heterogeneity among genetically similar cells. Specifically, this approach provides an analytical tool for quantitatively investigating LD morphology in live cells in situ. With this high-throughput, high-resolution, and label-free method, we found that LD growth dynamics showed considerable cell to cell variation. Lipid accumulation in nonadipocyte cells is mainly reflected in the increase of LD number, as opposed to an increase in their size or lipid concentration. Our method allows statistical single-cell quantification of the LD distribution for further investigation of lipid metabolism and dynamic behavior, and also extends the possibility to couple with other "omics" technologies in the future. PMID:27041129

  13. Identification of Novel GPR55 Modulators Using Cell-Impedance-Based Label-Free Technology.

    Science.gov (United States)

    Morales, Paula; Whyte, Lauren S; Chicharro, Roberto; Gómez-Cañas, María; Pazos, M Ruth; Goya, Pilar; Irving, Andrew J; Fernández-Ruiz, Javier; Ross, Ruth A; Jagerovic, Nadine

    2016-03-10

    The orphan G protein-coupled receptor GPR55 has been proposed as a novel receptor of the endocannabinoid system. However, the validity of this categorization is still under debate mainly because of the lack of potent and selective agonists and antagonists of GPR55. Binding assays are not yet available for GPR55 screening, and discrepancies in GPR55 mediated signaling pathways have been reported. In this context, we have designed and synthesized novel GPR55 ligands based on a chromenopyrazole scaffold. Appraisal of GPR55 activity was accomplished using a label-free cell-impedance-based assay in hGPR55-HEK293 cells. The real-time impedance responses provided an integrative assessment of the cellular consequence to GPR55 stimulation taking into account the different possible signaling pathways. Potent GPR55 partial agonists (14b, 18b, 19b, 20b, and 21-24) have been identified; one of them (14b) being selective versus classical cannabinoid receptors. Upon antagonist treatment, chromenopyrazoles 21-24 inhibited lysophosphatidylinositol (LPI) effect. One of these GPR55 antagonists (21) is fully selective versus classic cannabinoid receptors. Compared to LPI, the predicted physicochemical parameters of the new compounds suggest a clear pharmacokinetic improvement.

  14. Identification of Novel GPR55 Modulators Using Cell-Impedance-Based Label-Free Technology.

    Science.gov (United States)

    Morales, Paula; Whyte, Lauren S; Chicharro, Roberto; Gómez-Cañas, María; Pazos, M Ruth; Goya, Pilar; Irving, Andrew J; Fernández-Ruiz, Javier; Ross, Ruth A; Jagerovic, Nadine

    2016-03-10

    The orphan G protein-coupled receptor GPR55 has been proposed as a novel receptor of the endocannabinoid system. However, the validity of this categorization is still under debate mainly because of the lack of potent and selective agonists and antagonists of GPR55. Binding assays are not yet available for GPR55 screening, and discrepancies in GPR55 mediated signaling pathways have been reported. In this context, we have designed and synthesized novel GPR55 ligands based on a chromenopyrazole scaffold. Appraisal of GPR55 activity was accomplished using a label-free cell-impedance-based assay in hGPR55-HEK293 cells. The real-time impedance responses provided an integrative assessment of the cellular consequence to GPR55 stimulation taking into account the different possible signaling pathways. Potent GPR55 partial agonists (14b, 18b, 19b, 20b, and 21-24) have been identified; one of them (14b) being selective versus classical cannabinoid receptors. Upon antagonist treatment, chromenopyrazoles 21-24 inhibited lysophosphatidylinositol (LPI) effect. One of these GPR55 antagonists (21) is fully selective versus classic cannabinoid receptors. Compared to LPI, the predicted physicochemical parameters of the new compounds suggest a clear pharmacokinetic improvement. PMID:26789378

  15. Imaging Alzheimer's disease-related protein aggregates in human cells using a selenium label

    International Nuclear Information System (INIS)

    The aberrant folding and subsequent aggregation of proteins and peptides is associated with a range of pathological conditions from the systemic amyloidoses to neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. While this link is well established there is a lack of understanding of the exact role protein aggregates play in disease pathogenesis. Part of the reason for this is that it has proved extremely challenging to characterise the localisation and structure of amyloid fibrils within the cellular environment due to a lack of contrast between the carbon rich protein aggregates and the carbon rich cell. We report a novel method for visualising Alzheimer's disease-related amyloid fibrils inside human cells without the use of invasive or unreliable stains or tags. The naturally occurring sulfur atom in the amyloid-β peptide is replaced with a selenium atom, a heavier element in the same group of the periodic table of elements. Using high angle annular dark field (HAADF) in a scanning transmission electron microscopy (STEM) the selenium-labelled aggregates can be identified within the cellular environment.

  16. Intrinsic indicator of photodamage during label-free multiphoton microscopy of cells and tissues.

    Directory of Open Access Journals (Sweden)

    Roberta Galli

    Full Text Available Multiphoton imaging has evolved as an indispensable tool in cell biology and holds prospects for clinical applications. When addressing endogenous signals such as coherent anti-Stokes Raman scattering (CARS or second harmonic generation, it requires intense laser irradiation that may cause photodamage. We report that increasing endogenous fluorescence signal upon multiphoton imaging constitutes a marker of photodamage. The effect was studied on mouse brain in vivo and ex vivo, on ex vivo human brain tissue samples, as well as on glioblastoma cells in vitro, demonstrating that this phenomenon is common to a variety of different systems, both ex vivo and in vivo. CARS microscopy and vibrational spectroscopy were used to analyze the photodamage. The development of a standard easy-to-use model that employs rehydrated cryosections allowed the characterization of the irradiation-induced fluorescence and related it to nonlinear photodamage. In conclusion, the monitoring of endogenous two-photon excited fluorescence during label-free multiphoton microscopy enables to estimate damage thresholds ex vivo as well as detect photodamage during in vivo experiments.

  17. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics

    DEFF Research Database (Denmark)

    Ong, S.E.; Blagoev, B.; Kratchmarova, I.;

    2002-01-01

    . Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non......Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures......-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred...

  18. Determination of the internalization pathway of photoluminescent nanodiamonds in mammalian cells for biological labeling and optimization of the ?uorescent yield

    CERN Document Server

    Faklaris, Orestis; Irinopoulou, Theano; Tauc, Patrick; Girard, Hugues; Gesset, Celine; Senour, Mohamed; Thorel, Alain; Arnault, Jean-Charles; Boudou, Jean-Paul; Curmi, Patrick A; Treussart, François

    2009-01-01

    Diamond nanoparticles have been recently used as new ?uorescent labels in cells. Their emission relies on color centers embedded in the diamond matrix. In this work we compare the photoluminescence of a single color center embedded in a 30 nm diameter nanodiamond to that to a single dye molecule and demonstrate the perfect photostability of the color centers. We also compare the photoluminescence properties of nanodiamonds prepared under different conditions in order to ?nd the optimal parameters to achieve a high ?uorescence yield. We use these photoluminescent nanodiamonds for HeLa cell labeling and investigate their uptake mechanism. On the one hand by immunostaining the endocytotic vesicles and on the other by transmission electron microscopy observations we study the localization of nanodiamonds in cells. Moreover, by blocking selectively different endocytotic mechanisms we unravel their internalization pathway. We ?nd that nanodiamonds enter the cells by receptor-mediated endocytosis. The results of thi...

  19. Stem cell research in hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Chengyi SUN; Shi ZUO

    2008-01-01

    The traditional view that adult human liver tumors, mainly hepatocellular carcinoma (HCC), arise from mature cell types has been challenged in recent dec-ades. The results of several studies suggest that HCC can be derived from liver stem cells. There are four levels of cells in the liver stem cell lineage: hepatocytes, hepatic stem cells/oval cells, bone marrow stem cells and hepato-pancreas stem cells. However, whether HCC is resulted from the differentiation block of stem cells and, moreover, which liver stem cell lineage is the source cell of hepatocarcinogenesis remain controversial. In this review, we focus on the current status of liver stem cell research and their roles in carcinogenesis of HCC, in order to explore new approaches for stem cell therapy of HCC.

  20. α-Ketoacids as precursors for phenylalanine and tyrosine labelling in cell-based protein overexpression.

    Science.gov (United States)

    Lichtenecker, Roman J; Weinhäupl, Katharina; Schmid, Walther; Konrat, Robert

    2013-12-01

    (13)C-α-ketoacid metabolic precursors of phenylalanine and tyrosine effectively enter the metabolism of a protein overexpressing E. coli strain to label Phe- and Tyr-residues devoid of any cross-labelling. The methodology gives access to highly selective labelling patterns as valuable tools in protein NMR spectroscopy without the need of (15)N-chiral amino acid synthesis using organic chemistry.

  1. In vivo tracking of 111In-oxine labeled mesenchymal stem cells following infusion in patients with advanced cirrhosis

    International Nuclear Information System (INIS)

    Background: Several animal and few human studies suggest the beneficial role of bone marrow mesenchymal stem cells (MSCs) in liver cirrhosis. However, little is known about the fate of MSCs after infusion in cirrhotic patients. We evaluated stem cell biodistribution after peripheral infusion of MSCs in four cirrhotic patients. Methods: After three passages of MSCs, the patients received a total of 250-400x106 cells, of which only 50% of the cells were labeled. Specific activities of 0.21-0.67 MBq/106 cells were maintained for the injected labeled MSCs. Planar whole-body acquisitions (anterior/posterior projections) were acquired immediately following infusion as well as at 2 h, 4 h, 6 h, 24 h, 48 h, 7th and 10th days after cell infusion. Results: After intravenous infusion, the radioactivity was first observed to accumulate in the lungs. During the following hours to days, the radioactivity gradually increased in the liver and spleen, with spleen uptake exceeding that in the liver in all patients. Region-of-interest analysis showed that the percentage of cells homing to the liver (following decay and background corrections and geometric mean calculation) increased from 0.0%-2.8% at immediately post-infusion images to 13.0-17.4% in 10th-day post-infusion. Similarly, the residual activities in the spleen increased from 2.0%-10.2% at immediately post-infusion images to 30.1%-42.2% in 10th-day post-infusion. During the same period, the residual activities in the lungs decreased from 27.0-33.5% to 2.0-5.4%. Conclusion: The infusion of MSCs labeled with 111In-oxine through a peripheral vein is safe in cirrhosis. Cell labeling with 111In-oxine is a suitable method for tracking MSC distribution after infusion.

  2. Synergetic effect of functional cadmium–tellurium quantum dots conjugated with gambogic acid for HepG2 cell-labeling and proliferation inhibition

    Directory of Open Access Journals (Sweden)

    Xu P

    2013-09-01

    Full Text Available Peipei Xu,1 Jingyuan Li,2 Lixin Shi,3 Matthias Selke,3 Baoan Chen,4 Xuemei Wang5 1Department of Hematology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, People’s Republic of China; 2Laboratory Animal Center, Institute of Comparative Medicine, Nantong University, Nantong, People’s Republic of China; 3Department of Chemistry and Biochemistry, California State University – Los Angeles, Los Angeles, CA, USA; 4Department of Hematology, Zhongda Hospital, Southeast University, Nanjing, People’s Republic of China; 5State Key Lab of Bioelectronics (Chien-Shiung Wu Laboratory, Southeast University, Nanjing, People’s Republic of ChinaAbstract: We prepared and studied novel fluorescent nanocomposites based on gambogic acid (GA and cadmium–tellurium (CdTe quantum dots (CdTe QDs modified with cysteamine for purpose of cancer cell labeling and combined treatment. The nanocomposites were denoted as GA-CdTe. Characterization results indicated that the CdTe QDs can readily bind onto cell plasma membranes and then be internalized into cancer cells for real-time labeling and tracing of human liver hepatocellular carcinoma cell line (HepG2 cells. GA-CdTe significantly enhanced drug accumulation in HepG2 cells and inhibited cancer cell proliferation. GA-CdTe nanocomposites also improved the drug action of GA molecules in HepG2 cells and induced the G2/M phase arrest of the cancer cell cycle, promoting cell apoptosis. Given the sensitive, pH-triggered release of GA-CdTe, the side effects of GA anticancer agents on normal cells/tissues in the blood circulation markedly decreased. Efficient drug release and accumulation in target tumor cells were also facilitated. Thus, the fluorescent GA-CdTe offered a new strategy for potential multimode cancer therapy and provided new channels for research into naturally-active compounds extracted from traditional Chinese medicinal plants.Keywords: cadmium-tellurium quantum dots

  3. Huge Varicose Inferior Mesenteric Vein: an Unanticipated {sup 99m}Tc-labeled Red Blood Cell Scintigraphy Finding

    Energy Technology Data Exchange (ETDEWEB)

    Hoseinzadeh, Samaneh; Shafiei, Babak; Salehian, Mohamadtaghi; Neshandar Asli, Isa; Ghodoosi, Iraj [Shaheed Beheshti Medical University, Tehran (Iran, Islamic Republic of)

    2010-09-15

    Ectopic varices (EcV) are enlarged portosystemic venous collaterals, which usually develop secondary to portal hypertension (PHT). Mesocaval collateral vessels are unusual pathways to decompress the portal system. Here we report the case of a huge varicose inferior mesenteric vein (IMV) that drained into peri rectal collateral veins, demonstrated by {sup 99m}Tc-labeled red blood cell (RBC) scintigraphy performed for lower gastrointestinal (GI) bleeding in a 14-year-old girl. This case illustrates the crucial role of {sup 99m}Tc-labeled RBC scintigraphy for the diagnosis of rare ectopic lower GI varices.

  4. Targeting of 111In-Labeled Dendritic Cell Human Vaccines Improved by Reducing Number of Cells

    NARCIS (Netherlands)

    Aarntzen, E.H.J.G.; Srinivas, M.; Bonetto, F.J.; Cruz, L.J.; Verdijk, P.; Schreibelt, G.; Rakt, M.W.M.M. van de; Lesterhuis, W.J.; Riel, M. van; Punt, C.J.A.; Adema, G.J.; Heerschap, A.; Figdor, C.G.; Oyen, W.J.G.; Vries, I.J.M. de

    2013-01-01

    PURPOSE: Anticancer dendritic cell (DC) vaccines require the DCs to relocate to lymph nodes (LN) to trigger immune responses. However, these migration rates are typically very poor. Improving the targeting of ex vivo generated DCs to LNs might increase vaccine efficacy and reduce costs. We investiga

  5. Research perspectives and role of lactose uptake rate revealed by its study using 14C-labelled lactose in whey fermentation.

    Science.gov (United States)

    Golfinopoulos, Aristidis; Kopsahelis, Nikolaos; Tsaousi, Konstantina; Koutinas, Athanasios A; Soupioni, Magdalini

    2011-03-01

    The present investigation examines the effect of pH, temperature and cell concentration on lactose uptake rate, in relation with kinetics of whey fermentation using kefir and determines the optimum conditions of these parameters. Lactose uptake rate was measured by adding (14)C-labelled lactose in whey. The results reveal the role of lactose uptake rate, being the main factor that affects the rate of fermentation, in contrast to the activity of the enzymes involved in lactose bioconversion process. Lactose uptake rate results discussion showed that mainly Ca(2+) is responsible for the reduced whey fermentation rate in comparison with fermentations using synthetic media containing lactose. Likewise, the results draw up perspectives on whey fermentation research to improve whey fermentation rate. Those perspectives are research to remove Ca(2+) from whey, the use of nano and microtubular biopolymers and promoters such as γ-alumina pellets and volcan foaming rock kissiris in order to accelerate whey fermentation. PMID:21232943

  6. Correlation between the rate of bioreduction of nitroxide spin label by human tumor cells and their low-dose radiation response

    International Nuclear Information System (INIS)

    The authors discuss a correlation observed between the bioreduction of nitroxide spin label by four human tumor cell lines and a normal tissue fibroblast clone and their low-dose radiation response, specifically their DQ. In measurements of the bioreduction rate of several other cell lines, this correlation appears to persist. In order to define the mechanism of this correlation, they have begun by subtly altering the measurement conditions. The original conditions for measurement involved adding the spin label to cells whose culture medium had been changed (the label was added to the new medium). By delaying the addition of the label to the culture medium, they substantially reduced the variation of the bioreduction rate between the cell lines. This implies that the fresh medium provides a nonspecific irritant or disequilibrium to the cultured cell system to which they response variably by accelerating, among other things, the metabolic process responsible for spin label bioreduction

  7. The effect of 125I labeled anti-androgen receptor agent on the proliferation of prostate cancer cells

    International Nuclear Information System (INIS)

    Objective: Based on the previous experience of using anti-androgen receptor triple helix forming oligonucleotide (TFO) to inhibit proliferation of prostate cancer cells, a 125I labeled TFO was prepared and tested in this experiment as an androgen receptor targeted antigene radiotherapy. Methods: 125I-TFO was labeled through Iodogen and then transfected LNCaP prostate cancer cells via liposome. The unlabeled TFO, 125I, and naturally cultured cells served as controls. The cellular proliferation was detected by methyl thiazolium tetrazolium (MTT) method, the expression of androgen receptor gene was carried out by RT-PCR and immunohistochemical study. Results: The radiolabeling efficiency, radiochemical purity and specific activity of 125I-TFO were 63.7%, 95.6% and 80.1 kBq/μg, respectively. At the same TFO concentration, the androgen receptor expression level in 125I-TFO treated cells was markedly lower than that of TFO group (P125I-TFO on cellular proliferation was significantly higher (P< 0.01). Conclusion: The inhibitory effect on androgen receptor expression and cell proliferation of prostate cancer cells of antigene therapy with radio-labeled TFO were significantly more obvious than that of classical antigene therapy. (authors)

  8. Adaptation of a Commonly Used, Chemically Defined Medium for Human Embryonic Stem Cells to Stable Isotope Labeling with Amino Acids in Cell Culture

    DEFF Research Database (Denmark)

    Liberski, A. R.; Al-Noubi, M. N.; Rahman, Z. H.;

    2013-01-01

    rarely employed in the context of complex culturing conditions as those required for human embryonic stem cells (hESCs). Classic hESC culture is based on the use of mouse embryonic fibroblasts (MEFs) as a feeder layer, and as a result, possible xenogeneic contamination, contribution of unlabeled amino......Metabolic labeling with stable isotopes is a prominent technique for comparative quantitative proteomics, and stable isotope labeling with amino acids in cell culture (SILAC) is the most commonly used approach. SILAC is, however, traditionally limited to simple tissue culture regimens and only...... developed by Ludwig et al. and commercially available as mTeSR1 [mTeSR1 is a trade mark of WiCell (Madison, WI) licensed to STEMCELL Technologies (Vancouver, Canada)]. This medium, together with adjustments to the culturing protocol, facilitates reproducible labeling that is easily scalable to the protein...

  9. Fluorescence-Activated Cell Sorting of EGFP-Labeled Neural Crest Cells From Murine Embryonic Craniofacial Tissue

    Directory of Open Access Journals (Sweden)

    Saurabh Singh

    2005-01-01

    Full Text Available During the early stages of embryogenesis, pluripotent neural crest cells (NCC are known to migrate from the neural folds to populate multiple target sites in the embryo where they differentiate into various derivatives, including cartilage, bone, connective tissue, melanocytes, glia, and neurons of the peripheral nervous system. The ability to obtain pure NCC populations is essential to enable molecular analyses of neural crest induction, migration, and/or differentiation. Crossing Wnt1-Cre and Z/EG transgenic mouse lines resulted in offspring in which the Wnt1-Cre transgene activated permanent EGFP expression only in NCC. The present report demonstrates a flow cytometric method to sort and isolate populations of EGFP-labeled NCC. The identity of the sorted neural crest cells was confirmed by assaying expression of known marker genes by TaqMan Quantitative Real-Time Polymerase Chain Reaction (QRT-PCR. The molecular strategy described in this report provides a means to extract intact RNA from a pure population of NCC thus enabling analysis of gene expression in a defined population of embryonic precursor cells critical to development.

  10. Label-free isolation and enrichment of cells through contactless dielectrophoresis.

    Science.gov (United States)

    Elvington, Elizabeth S; Salmanzadeh, Alireza; Stremler, Mark A; Davalos, Rafael V

    2013-09-03

    Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to direct the motion of microparticles in a surface marker-independent manner. Traditionally, DEP devices include planar metallic electrodes patterned in the sample channel. This approach can be expensive and requires a specialized cleanroom environment. Recently, a contact-free approach called contactless dielectrophoresis (cDEP) has been developed. This method utilizes the classic principle of DEP while avoiding direct contact between electrodes and sample by patterning fluidic electrodes and a sample channel from a single polydimethylsiloxane (PDMS) substrate, and has application as a rapid microfluidic strategy designed to sort and enrich microparticles. Unique to this method is that the electric field is generated via fluidic electrode channels containing a highly conductive fluid, which are separated from the sample channel by a thin insulating barrier. Because metal electrodes do not directly contact the sample, electrolysis, electrode delamination, and sample contamination are avoided. Additionally, this enables an inexpensive and simple fabrication process. cDEP is thus well-suited for manipulating sensitive biological particles. The dielectrophoretic force acting upon the particles depends not only upon spatial gradients of the electric field generated by customizable design of the device geometry, but the intrinsic biophysical properties of the cell. As such, cDEP is a label-free technique that avoids depending upon surface-expressed molecular biomarkers that may be variably expressed within a population, while still allowing characterization, enrichment, and sorting of bioparticles. Here, we demonstrate the basics of fabrication and experimentation using cDEP. We explain the simple preparation of a cDEP chip using soft lithography techniques. We discuss the experimental procedure for characterizing

  11. Infrared fluorescent protein 1.4 genetic labeling tracks engrafted cardiac progenitor cells in mouse ischemic hearts.

    Directory of Open Access Journals (Sweden)

    Lijuan Chen

    Full Text Available Stem cell therapy has a potential for regenerating damaged myocardium. However, a key obstacle to cell therapy's success is the loss of engrafted cells due to apoptosis or necrosis in the ischemic myocardium. While many strategies have been developed to improve engrafted cell survival, tools to evaluate cell efficacy within the body are limited. Traditional genetic labeling tools, such as GFP-like fluorescent proteins (eGFP, DsRed, mCherry, have limited penetration depths in vivo due to tissue scattering and absorption. To circumvent these limitations, a near-infrared fluorescent mutant of the DrBphP bacteriophytochrome from Deinococcus radiodurans, IFP1.4, was developed for in vivo imaging, but it has yet to be used for in vivo stem/progenitor cell tracking. In this study, we incorporated IFP1.4 into mouse cardiac progenitor cells (CPCs by a lentiviral vector. Live IFP1.4-labeled CPCs were imaged by their near-infrared fluorescence (NIRF using an Odyssey scanner following overnight incubation with biliverdin. A significant linear correlation was observed between the amount of cells and NIRF signal intensity in in vitro studies. Lentiviral mediated IFP1.4 gene labeling is stable, and does not impact the apoptosis and cardiac differentiation of CPC. To assess efficacy of our model for engrafted cells in vivo, IFP1.4-labeled CPCs were intramyocardially injected into infarcted hearts. NIRF signals were collected at 1-day, 7-days, and 14-days post-injection using the Kodak in vivo multispectral imaging system. Strong NIRF signals from engrafted cells were imaged 1 day after injection. At 1 week after injection, 70% of the NIRF signal was lost when compared to the intensity of the day 1 signal. The data collected 2 weeks following transplantation showed an 88% decrease when compared to day 1. Our studies have shown that IFP1.4 gene labeling can be used to track the viability of transplanted cells in vivo.

  12. DNA labeling in vivo: Quantification of epidermal stem cell chromatin content in whole mouse hair follicles using Fiji image processing software

    OpenAIRE

    Carrasco, Elisa; Calvo, María I.; Espada, Jesús

    2014-01-01

    DNA labeling in vivo using nucleoside analogues is a current experimental approach to determine cell proliferation rates in cell cultures and tissues. It has also been successfully used to localize adult stem cell niches through the identification of nucleoside label-retaining cells (LRC) in long-term experiments. A major hindrance of this methodology relies on the selection of adequate procedures to quantify the nucleoside analogue content from image data files. Here we propose a simple proc...

  13. The study of optimal condition of SPIO labeling human lung adenocarcinoma cell line (SPC-A-1)

    Science.gov (United States)

    Yu, Ming-xi; Chen, Wen-li; Zhou, Quan; Xing, Da; Tang, Yong-hong

    2008-02-01

    Propose: To study the optimal concentration and time of incubation of human lung adenocarcinoma cell line (SPC-A-1) labeled with superparamagnetic iron oxide (SPIO) particles in vitro. Methods: Human lung adenocarcinoma cell line (SPC-A-1) was cultured with different concenration of SPIO and different time of incubation (labeled with media containing Fe-PLL: 25μg /mL, 100μg /mL, and 200 μg /mL, and for 30min, 90min, 180min. The phagocytosis of the cells was observed by laser scanning confocal microscopy (LSCM) to determine particle uptake and their distribution in cells. Results: Human lung adenocarcinoma cells(SPC-A-1) have taken up a large amount of SPIO particles within the first 3h. Conclusion: In this study, the concentration of iron with 25μg/ml SPIO and time of incubation for 30min is the optimal condition for labeling the SPC-A-1 with SPIO.

  14. In vivo MRI tracking of iron oxide nanoparticle-labeled human mesenchymal stem cells in limb ischemia

    Directory of Open Access Journals (Sweden)

    Li XX

    2013-03-01

    Full Text Available Xiang-Xiang Li,1,2,* Kang-An Li,3,* Jin-Bao Qin,1,2 Kai-Chuang Ye,1,2 Xin-Rui Yang,1,2 Wei-Min Li,1,2 Qing-Song Xie,4 Mi-Er Jiang,1,2 Gui-Xiang Zhang,3 Xin-Wu Lu1,21Department of Vascular Surgery, Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University, School of Medicine, 2Vascular Center, Shanghai JiaoTong University, 3Department of Radiology, Shanghai First People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China; 4Department of Neurosurgery, Cixi Municipal People's Hospital, Zhejiang Province, People's Republic of China *These authors contributed equally to this work Background: Stem cell transplantation has been investigated for repairing damaged tissues in various injury models. Monitoring the safety and fate of transplanted cells using noninvasive methods is important to advance this technique into clinical applications. Methods: In this study, lower-limb ischemia models were generated in nude mice by femoral artery ligation. As negative-contrast agents, positively charged magnetic iron oxide nanoparticles (aminopropyltriethoxysilane-coated Fe2O3 were investigated in terms of in vitro labeling efficiency, effects on human mesenchymal stromal cell (hMSC proliferation, and in vivo magnetic resonance imaging (MRI visualization. Ultimately, the mice were sacrificed for histological analysis three weeks after transplantation. Results: With efficient labeling, aminopropyltriethoxysilane-modified magnetic iron oxide nanoparticles (APTS-MNPs did not significantly affect hMSC proliferation. In vivo, APTS-MNP-labeled hMSCs could be monitored by clinical 3 Tesla MRI for at least three weeks. Histological examination detected numerous migrated Prussian blue-positive cells, which was consistent with the magnetic resonance images. Some migrated Prussian blue-positive cells were positive for mature endothelial cell markers of von Willebrand factor and anti-human proliferating cell

  15. Efficient labeling in vitro with non-ionic gadolinium magnetic resonance imaging contrast agent and fluorescent transfection agent in bone marrow stromal cells of neonatal rats.

    Science.gov (United States)

    Li, Ying-Qin; Tang, Ying; Fu, Rao; Meng, Qiu-Hua; Zhou, Xue; Ling, Ze-Min; Cheng, Xiao; Tian, Su-Wei; Wang, Guo-Jie; Liu, Xue-Guo; Zhou, Li-Hua

    2015-07-01

    Although studies have been undertaken on gadolinium labeling-based molecular imaging in magnetic resonance imaging (MRI), the use of non-ionic gadolinium in the tracking of stem cells remains uncommon. To investigate the efficiency in tracking of stem cells with non-ionic gadolinium as an MRI contrast agent, a rhodamine-conjugated fluorescent reagent was used to label bone marrow stromal cells (BMSCs) of neonatal rats in vitro, and MRI scanning was undertaken. The fluorescent-conjugated cell uptake reagents were able to deliver gadodiamide into BMSCs, and cell uptake was verified using flow cytometry. In addition, the labeled stem cells with paramagnetic contrast medium remained detectable by an MRI monitor for a minimum of 28 days. The present study suggested that this method can be applied efficiently and safely for the labeling and tracking of bone marrow stromal cells in neonatal rats.

  16. Autologous Bone Marrow Mononuclear Cell Therapy for Autism: An Open Label Proof of Concept Study

    Directory of Open Access Journals (Sweden)

    Alok Sharma

    2013-01-01

    Full Text Available Cellular therapy is an emerging therapeutic modality with a great potential for the treatment of autism. Recent findings show that the major underlying pathogenetic mechanisms of autism are hypoperfusion and immune alterations in the brain. So conceptually, cellular therapy which facilitates counteractive processes of improving perfusion by angiogenesis and balancing inflammation by immune regulation would exhibit beneficial clinical effects in patients with autism. This is an open label proof of concept study of autologous bone marrow mononuclear cells (BMMNCs intrathecal transplantation in 32 patients with autism followed by multidisciplinary therapies. All patients were followed up for 26 months (mean 12.7. Outcome measures used were ISAA, CGI, and FIM/Wee-FIM scales. Positron Emission Tomography-Computed Tomography (PET-CT scan recorded objective changes. Out of 32 patients, a total of 29 (91% patients improved on total ISAA scores and 20 patients (62% showed decreased severity on CGI-I. The difference between pre- and postscores was statistically significant (P<0.001 on Wilcoxon matched-pairs signed rank test. On CGI-II 96% of patients showed global improvement. The efficacy was measured on CGI-III efficacy index. Few adverse events including seizures in three patients were controlled with medications. The encouraging results of this leading clinical study provide future directions for application of cellular therapy in autism.

  17. Labeling of Chromosomes in Cell Development and the Appearance of Monozygotic Twins

    Directory of Open Access Journals (Sweden)

    Carol Jim

    2015-01-01

    Full Text Available Understanding the mechanism behind the structure of the internal cellular clock can lead to advances in the knowledge of origins of pairs of monozygotic twins and higher order multiples as well as other biological phenomena. To gain insight into this mechanism, we analyze possible cell labeling schemes that model an organism’s development. Our findings lead us to predict that monozygotic quadruplets are not quadruplets in the traditional sense but rather two pairs of monozygotic twins where the pairs slightly differ—a situation we coin quadruplet twins. From the considered model, the probability of monozygotic twins is found to be 1/2K, and we discover that the probability of monozygotic quadruplets, or triplets as in the case of the death of an embryo, is 1/8K, where K is a species-specific integer representing the number of pairs of homologous chromosomes. The parameter K may determine cancerization with a probability threshold that is approximately inversely proportional to the Hayflick limit. Exposure to some cancerization factors such as small levels of ionizing radiation and chemical pollution may not produce cancer.

  18. Sickle cell anemia: reference values of cerebral blood flow determined by continuous arterial spin labeling MRI.

    Science.gov (United States)

    Arkuszewski, M; Krejza, J; Chen, R; Melhem, E R

    2013-04-01

    Sickle cell anemia (SCA) is a chronic illness associated with progressive deterioration in patients' quality of life. The major complications of SCA are cerebrovascular accidents (CVA) such as asymptomatic cerebral infarct or overt stroke. The risk of CVA may be related to chronic disturbances in cerebral blood flow (CBF), but the thresholds of "normal" steady-state CBF are not well established. The reference tolerance limits of CBF can be useful to estimate the risk of CVA in asymptomatic children with SCA, who are negative for hyperemia or evidence of arterial narrowing. Continuous arterial spin labeling (CASL) MR perfusion allows for non-invasive quantification of global and regional CBF. To establish such reference tolerance limits we performed CASL MR examinations on a 3-Tesla MR scanner in a carefully selected cohort of 42 children with SCA (mean age, 8.1±3.3 years; range limits, 2.3-14.4 years; 24 females), who were not on chronic transfusion therapy, had no history of overt stroke or transient ischemic attack, were free of signs and symptoms of focal vascular territory ischemic brain injury, did not have intracranial arterial narrowing on MR angiography and were at low risk for stroke as determined by transcranial Doppler ultrasonography.

  19. A novel self-powered and sensitive label-free DNA biosensor in microbial fuel cell.

    Science.gov (United States)

    Asghary, Maryam; Raoof, Jahan Bakhsh; Rahimnejad, Mostafa; Ojani, Reza

    2016-08-15

    In this work, a novel self-powered, sensitive, low-cost, and label-free DNA biosensor is reported by applying a two-chambered microbial fuel cell (MFC) as a power supply. A graphite electrode and an Au nanoparticles modified graphite electrode (AuNP/graphite electrode) were used as anode and cathode in the MFC system, respectively. The active biocatalyst in the anodic chamber was a mixed culture of microorganisms. The sensing element of the biosensor was fabricated by the well-known Au-thiol binding the ssDNA probe on the surface of an AuNP/graphite cathode. Electrons produced by microorganisms were transported from the anode to the cathode through an external circuit, which could be detected by the terminal multi-meter detector. The difference between power densities of the ssDNA probe modified cathode in the absence and presence of complementary sequence served as the detection signal of the DNA hybridization with detection limit of 3.1nM. Thereafter, this biosensor was employed for diagnosis and determination of complementary sequence in a human serum sample. The hybridization specificity studies further revealed that the developed DNA biosensor could distinguish fully complementary sequences from one-base mismatched and non-complementary sequences. PMID:27085948

  20. Preparation of Fluorescent Dye-Doped Biocompatible Nanoparticles for Cell Labeling.

    Science.gov (United States)

    Wang, Xiaohui; Peng, Hongshang; Huang, Shihua; You, Fangtian

    2016-04-01

    In this paper, we report a series of fluorescent biocompatible nanoparticles (NPs), prepared by a facile reprecipitation-encapsulation method, for cellular labeling. The as-prepared NPs exhibit a narrow size distribution of 70-110 nm, and a core-shell structure comprised of a hybrid core doped with different dyes and a poly-L-lysine (PLL) shell. With coumarin 6, nile red, and meso- tetraphenylporphyrin as the imaging agents, the fluorescent NPs gave green, orange, and red emissions respectively. Due to the positively charged PLL shell, the fluorescent NPs exhibit neglected cytotoxicity and efficient cellular uptake. After incubation with living cells, the results obtained by laser confocal microscope from green, orange, and red channels all clearly show that the fluores- cent NPs are inhomogenously localized inside the cytoplasm without penetrating into the nucleus. Since such PLL-modified NPs can encapsulate other hydrophobic dyes, a wide spectrum of nanoimaging agents is thus expected. Furthermore, the surface amino groups on the PLL shell afford an anchoring site for further bioconjugation, and targeted imaging is also very promising. PMID:27451673

  1. The research analysis of the green label's impact on the consumer purchase behavior

    OpenAIRE

    Xu, Yan

    2013-01-01

    Nowadays, environmental protection has drawn more and more people’s attention. Therefore, many companies take Green Marketing as an important marketing strategy in order to sustain their own advantages in the fiercely competitive market environment. They take use of environmentally protective advertisements, eco-brand and eco-label to remind people’s perception and awareness of green products, make it easier to define the attributes and characteristics of green products, and then consequently...

  2. Ethical Issues in Stem Cell Research

    OpenAIRE

    Lo, Bernard; Parham, Lindsay

    2009-01-01

    Stem cell research offers great promise for understanding basic mechanisms of human development and differentiation, as well as the hope for new treatments for diseases such as diabetes, spinal cord injury, Parkinson’s disease, and myocardial infarction. However, human stem cell (hSC) research also raises sharp ethical and political controversies. The derivation of pluripotent stem cell lines from oocytes and embryos is fraught with disputes about the onset of human personhood. The reprogramm...

  3. Estimations of the DNA Synthesis Rate of Bone Marrow Cells after Administration of Labelled Thymidine In Vitro

    International Nuclear Information System (INIS)

    Bone marrow cells are incubated with labelled thymidine under varying in vitro conditions. The incorporation rate of labelled thymidine into DNA is influenced by the condition and duration of. the in vitro incubation. Similar influences operate on the pool size of labelled thymidine phosphates. Up to concentrations of 10-6 M thymidine in the incubation medium there is a linear relation of thymidine concentration and thymidine incorporation into DNA. Concentrations of thymidine exceeding 10-6 M lead to increasing inhibition of the thymidine kinase. The endogenous formation of thymidylate cannot be inhibited entirely by exogenous thymidine supply. Consequently, determinations of the DNA synthesis rate from the incorporated amount of labelled thymidine have to be corrected for the respective endogenous thymidylate contribution. A better procedure is to block the formation of endogenous thymidylate by means of amethopterin. Standard conditions are described, under which an undisturbed synthesis of DNA thymine from exogenous thymidine only takes place. Determinations can be performed by means of autoradiographic or biochemical techniques. By application of the semi-automatic grain counting technique, after sufficient autoradiographic standardization, evaluations of DNA synthesis rates and DNA synthesis times of different cell types in the bone marrow become practicable. (author)

  4. 111In oxine labeled red cells for detection of simulated lower gastrointestinal bleeding in an animal model

    International Nuclear Information System (INIS)

    111In oxine in vitro labeled red cells were evaluated in rabbits for the ability to detect gastrointestinal (Gl) bleeding. A mean labeling efficiency of 81% (+- 15.5%) was achieved. Biodistribution and translocation data demonstrated 81% of the activity within the blood pool at four hours after intravenous injection, falling to 29% by 72 hours. Peak urine excretion occurred after 60 to 150 minutes. Normal Gl excretion was less than 1% over 72 hours. Simulated lower Gl bleeding was imaged at 4, 12, and 72 hours, and amounts as small as 2 ml (1% blood volume) were seen. In rabbits the total body dose of injected 111In is 0.15 mGy/MBq (0.56 rad/mCi), and the critical organ is the spleen, which received 0.49 mGy/MBq (1.82 rad/mCi). 111In oxine labeled red cells provide a sustained blood pool label wthout significant accumulation in the Gl tract, and may have a potential use in the detection of intermittent Gl bleeding in humans

  5. Specific detection of CD133-positive tumor cells with iron oxide nanoparticles labeling using noninvasive molecular magnetic resonance imaging

    Directory of Open Access Journals (Sweden)

    Chen YW

    2015-11-01

    Full Text Available Ya-Wen Chen,1,2 Gunn-Guang Liou,3 Huay-Ben Pan,4,5 Hui-Hwa Tseng,5,6 Yu-Ting Hung,4 Chen-Pin Chou4,5,7,8 1National Institute of Cancer Research, National Health Research Institutes, Miaoli, 2Graduate Institute of Basic Medical Science, China Medical University, Taichung, 3Institute of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli, 4Department of Radiology, Kaohsiung Veterans General Hospital, Kaohsiung, 5School of Medicine, National Yang-Ming University, Taipei, 6Department of Pathology, Kaohsiung Veterans General Hospital, Kaohsiung, 7Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, 8School of Medicine, National Defense Medical Center, Taipei, Taiwan Background: The use of ultrasmall superparamagnetic iron oxide (USPIO nanoparticles to visualize cells has been applied clinically, showing the potential for monitoring cells in vivo with magnetic resonance imaging (MRI. USPIO conjugated with anti-CD133 antibodies (USPIO-CD133 Ab that recognize the CD133 molecule, a cancer stem cell marker in a variety of cancers, was studied as a novel and potent agent for MRI contrast enhancement of tumor cells. Materials and methods: Anti-CD133 antibodies were used to conjugate with USPIO via interaction of streptavidin and biotin for in vivo labeling of CD133-positive cells in xenografted tumors and N-ethyl-N-nitrosourea (ENU-induced brain tumors. The specific binding of USPIO-CD133 Ab to CD133-positive tumor cells was subsequently detected by Prussian blue staining and MRI with T2-weighted, gradient echo and multiple echo recombined gradient echo images. In addition, the cellular toxicity of USPIO-CD133 Ab was determined by analyzing cell proliferation, apoptosis, and reactive oxygen species production. Results: USPIO-CD133 Ab specifically recognizes in vitro and labels CD133-positive cells, as validated using Prussian blue staining and MRI. The assays of cell proliferation, apoptosis, and

  6. Isolation of Plant Nuclei at Defined Cell Cycle Stages Using EdU Labeling and Flow Cytometry.

    Science.gov (United States)

    Wear, Emily E; Concia, Lorenzo; Brooks, Ashley M; Markham, Emily A; Lee, Tae-Jin; Allen, George C; Thompson, William F; Hanley-Bowdoin, Linda

    2016-01-01

    5-Ethynyl-2'-deoxyuridine (EdU) is a nucleoside analog of thymidine that can be rapidly incorporated into replicating DNA in vivo and, subsequently, detected by using "click" chemistry to couple its terminal alkyne group to fluorescent azides such as Alexa Fluor 488. Recently, EdU incorporation followed by coupling with a fluorophore has been used to visualize newly synthesized DNA in a wide range of plant species. One particularly useful application is in flow cytometry, where two-parameter sorting can be employed to analyze different phases of the cell cycle, as defined both by total DNA content and the amount of EdU pulse-labeled DNA. This approach allows analysis of the cell cycle without the need for synchronous cell populations, which can be difficult to obtain in many plant systems. The approach presented here, which was developed for fixed, EdU-labeled nuclei, can be used to prepare analytical profiles as well as to make highly purified preparations of G1, S, or G2/M phase nuclei for molecular or biochemical analysis. We present protocols for EdU pulse labeling, tissue fixation and harvesting, nuclei preparation, and flow sorting. Although developed for Arabidopsis suspension cells and maize root tips, these protocols should be modifiable to many other plant systems. PMID:26659955

  7. Limited value of technetium 99m-labeled red cell scintigraphy in localization of lower gastrointestinal bleeding

    International Nuclear Information System (INIS)

    The aim of this study was to assess the accuracy of technetium 99m-labeled red cell scintigraphy in localizing the site of lower gastrointestinal bleeding. The outcome of 203 patients undergoing technetium 99m-labeled red cell scintigraphy was reviewed, and the scan result was compared with the true site of bleeding. The true site of bleeding was determined by other methods including angiography and surgical pathology. Fifty-two scans (26%) were positive and indicated a specific site of bleeding. A definitive bleeding site was identified in 22 patients by other means and correlated with the technetium scan in only 9 cases. The nuclear scan was incorrect in the remaining 13 cases, implying a localization error of 25% (13 of 52). A subgroup of 19 patients with a positive scan underwent a surgical procedure directed by the nuclear scan. Eight of these 12 patients had incorrect surgical procedures based upon findings of more definitive tests, indicating a surgical error of 42% (8 of 19). We conclude that the technetium 99m-labeled red cell scan's ability to accurately localize the site of lower gastrointestinal bleeding is limited. Furthermore, performing a surgical procedure that relies exclusively on localization by red cell scintigraphy will produce an undesirable result in at least 42% of patients

  8. Limited value of technetium 99m-labeled red cell scintigraphy in localization of lower gastrointestinal bleeding

    Energy Technology Data Exchange (ETDEWEB)

    Hunter, J.M.; Pezim, M.E. (Univ. of British Columbia, Vancouver (Canada))

    1990-05-01

    The aim of this study was to assess the accuracy of technetium 99m-labeled red cell scintigraphy in localizing the site of lower gastrointestinal bleeding. The outcome of 203 patients undergoing technetium 99m-labeled red cell scintigraphy was reviewed, and the scan result was compared with the true site of bleeding. The true site of bleeding was determined by other methods including angiography and surgical pathology. Fifty-two scans (26%) were positive and indicated a specific site of bleeding. A definitive bleeding site was identified in 22 patients by other means and correlated with the technetium scan in only 9 cases. The nuclear scan was incorrect in the remaining 13 cases, implying a localization error of 25% (13 of 52). A subgroup of 19 patients with a positive scan underwent a surgical procedure directed by the nuclear scan. Eight of these 12 patients had incorrect surgical procedures based upon findings of more definitive tests, indicating a surgical error of 42% (8 of 19). We conclude that the technetium 99m-labeled red cell scan's ability to accurately localize the site of lower gastrointestinal bleeding is limited. Furthermore, performing a surgical procedure that relies exclusively on localization by red cell scintigraphy will produce an undesirable result in at least 42% of patients.

  9. Human amniotic fluid stem cells labeled with up-conversion nanoparticles for imaging-monitored repairing of acute lung injury.

    Science.gov (United States)

    Xu, Yunyun; Xiang, Jian; Zhao, He; Liang, Hansi; Huang, Jie; Li, Yan; Pan, Jian; Zhou, Huiting; Zhang, Xueguang; Wang, Jiang Huai; Liu, Zhuang; Wang, Jian

    2016-09-01

    Human amniotic fluid stem (hAFS) cells have generated a great deal of excitement in cell-based therapies and regenerative medicine. Here, we examined the effect of hAFS cells labeled with dual-polymer-coated UCNP-PEG-PEI nanoparticles in a murine model of acute lung injury (ALI). We observed hAFS cells migration to the lung using highly sensitive in vivo upconversion luminescence (UCL) imaging. We demonstrated that hAFS cells remained viable and retained their ability to differentiate even after UCNP-PEG-PEI labeling. More importantly, hAFS cells displayed remarkable positive effects on ALI-damaged lung tissue repair compared with mouse bone marrow mesenchymal stem cells (mBMSCs), which include recovery of the integrity of alveolar-capillary membrane, attenuation of transepithelial leukocyte and neutrophil migration, and down-regulation of proinflammatory cytokine and chemokine expression. Our work highlights a promising role for imaging-guided hAFS cell-based therapy in ALI. PMID:27244692

  10. Breast tumors in PyMT transgenic mice expressing mitochondrial catalase have decreased labeling for macrophages and endothelial cells

    Directory of Open Access Journals (Sweden)

    Sy Fatemie

    2012-05-01

    Full Text Available We show by immunohistochemical labeling that prominent cell types in the tumor microenvironment of PyMT transgenic mice are tumor-associated macrophages (TAMs and endothelial cells, and that both populations are decreased in the presence of mitochondrial targeted catalase (mCAT. This observation suggests that mitochondrial ROS can drive tumor invasiveness in conjunction with the presence of TAMs and increased angiogenesis. Since primary PyMT tumor cells expressing mCAT undergo increased apoptosis, mitochondrial antioxidants might be attractive anti-tumor agents.

  11. Quantitative Proteomic Analysis of Bromotetrandrine and Tetrandrine in K562 Cell Line Using 18O-labeling Method

    Institute of Scientific and Technical Information of China (English)

    TAN Ying; GE Zhi-qiang; LIU Chang-xiao

    2012-01-01

    Objective To compare quantitative proteomic analysis of bromotetrandrine (W198) which was a Class Ⅰ new antitumor drug in China and tetrandrine (Tet) in K562 cell line using 18O-labeling method.Methods To illustrate its mechanism,a shotgun quantitative proteomic strategy employing 2D LC-MS-MS and trypsin catalyzed 18O-labeling quantification was carried out in this study.Compared to normal chronic leukemia cell line K562 and K562 induced by Tet,the proteomic changes of K562 induced by W198 were investigated.In order to validate the quantitation by the 18O-labeling,the analysis was done on an equivalent sample composed of the same amount of labeled and unlabeled proteins from normally cultured cells to act as a reference to the comparative sample.Results A threshold of ± 2-fold change for deciding whether a protein concentration was changed was settled for the following experiments.Comparing the 105 identified soluble proteins' expression levels of the apoptosis starting up K562 cells after W198 induction with the normally cultured cells,16 proteins were found with significantly altered expression levels after W198 treatment.Eight proteins were up-expressed including HMGB2,peroxiredoxin-2,and eIF4A-I,etc.Eight proteins were down-expressed including TCP-1,GRP94,GST-π,and SFGHs,etc.Compared to K562 induced by Tet,eight proteins of K562 were found with significantly altered expression levels after W198 treatment.Five proteins were up-expressed including HSP 90-β and 40S ribosomal protein S15a,etc.Three proteins were down-expressed including phosphoglycerate kinase 1,isoform 5 of interleukin enhancer-binding factor 3,etc.Conclusion The 18O-labeling MS-MS-based method is ideal as a discovery tool,but it is not suitable for validation using a large number of samples.Other more effective methods,such as Western blotting should be used for further validation of candidate cancer proteins discovered from 18O-labeling samples.In total,105 soluble proteins were discovered

  12. Peptide Biosynthesis with Stable Isotope Labeling from a Cell-free Expression System for Targeted Proteomics with Absolute Quantification.

    Science.gov (United States)

    Xian, Feng; Zi, Jin; Wang, Quanhui; Lou, Xiaomin; Sun, Haidan; Lin, Liang; Hou, Guixue; Rao, Weiqiao; Yin, Changcheng; Wu, Lin; Li, Shuwei; Liu, Siqi

    2016-08-01

    Because of its specificity and sensitivity, targeted proteomics using mass spectrometry for multiple reaction monitoring is a powerful tool to detect and quantify pre-selected peptides from a complex background and facilitates the absolute quantification of peptides using isotope-labeled forms as internal standards. How to generate isotope-labeled peptides remains an urgent challenge for accurately quantitative targeted proteomics on a large scale. Herein, we propose that isotope-labeled peptides fused with a quantitative tag could be synthesized through an expression system in vitro, and the homemade peptides could be enriched by magnetic beads with tag-affinity and globally quantified based on the corresponding multiple reaction monitoring signals provided by the fused tag. An Escherichia coli cell-free protein expression system, protein synthesis using recombinant elements, was adopted for the synthesis of isotope-labeled peptides fused with Strep-tag. Through a series of optimizations, we enabled efficient expression of the labeled peptides such that, after Strep-Tactin affinity enrichment, the peptide yield was acceptable in scale for quantification, and the peptides could be completely digested by trypsin to release the Strep-tag for quantification. Moreover, these recombinant peptides could be employed in the same way as synthetic peptides for multiple reaction monitoring applications and are likely more economical and useful in a laboratory for the scale of targeted proteomics. As an application, we synthesized four isotope-labeled glutathione S-transferase (GST) peptides and added them to mouse sera pre-treated with GST affinity resin as internal standards. A quantitative assay of the synthesized GST peptides confirmed the absolute GST quantification in mouse sera to be measurable and reproducible. PMID:27234506

  13. Increased Specific Labeling of INS-1 Pancreatic Beta-Cell by Using RIP-Driven Cre Mutants with Reduced Activity.

    Directory of Open Access Journals (Sweden)

    Gen-cheng Gong

    Full Text Available Ectopically expressed Cre recombinase in extrapancreatic tissues in RIP-Cre mice has been well documented. The objective of this study was to find a simple solution that allows for improved beta-cell specific targeting. To this end, the RIP-Cre and reporter CMV-loxP-DsRed-loxP-EGFP expression cassettes were configurated into a one-plasmid and two-plasmid systems, which labeled approximately 80% insulin-positive INS-1 cells after 48 h transfection. However, off-target labeling was robustly found in more than 15% insulin-negative Ad293 cells. When an IRES element was inserted in front of Cre to reduce the translation efficiency, the ratio of recombination between INS-1 and Ad293 cells increased 3-4-fold. Further, a series of Cre mutants were generated by site-directed mutagenesis. When one of the mutants, Cre(H289P in both configurations, was used in the experiment, the percentage of recombination dropped to background levels in a number of insulin-negative cell lines, but decreased only slightly in INS-1 cells. Consistently, DNA substrate digestion assay showed that the enzymatic activity of Cre(H289P was reduced by 30-fold as compared to that of wild-type. In this study, we reported the generation of constructs containing RIP and Cre mutants, which enabled enhanced beta-cell specific labeling in vitro. These tools could be invaluable for beta-cell targeting and to the study of islet development.

  14. Cell Wall Growth and Modulation Dynamics in a Model Unicellular Green Alga—Penium margaritaceum: Live Cell Labeling with Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    David S. Domozych

    2011-01-01

    Full Text Available Penium margaritaceum is a unicellular charophycean green alga that possesses cell wall polymers similar to those of land plants. Several wall macromolecules of this alga are recognized by monoclonal antibodies specific for wall polymer epitopes of land plants. Immunofluorescence protocols using these antibodies may be employed to label specific cell wall constituents of live cells. Fluorescent labeling persists for several days, and this attribute allows for tracing of wall epitopes in both long- and short-term studies of cell development. Quantitative analysis of surface area covered by cell wall polymers is also easily performed. We show that significant cell expansion caused by incubation of cells in low levels of osmotically active agents like mannitol, glucose, or sucrose results from the inability of cells to undergo cytokinesis but does not result in significant changes to the amount of new cell wall. We also demonstrate that cells can be maintained for long periods of time in culture medium supplemented with specific cell wall-degrading enzymes where notable changes to wall infrastructure occur. These results demonstrate the great potential value of Penium in elucidating fundamental events during cell wall synthesis and modulation in plant cells.

  15. Splenic scintigraphy using Tc-99m-labeled heat-denatured red blood cells in pediatric patients: concise communication

    Energy Technology Data Exchange (ETDEWEB)

    Ehrlich, C.P.; Papanicolaou, N.; Treves, S.; Hurwitz, R.A.; Richards, P.

    1982-03-01

    Ten children underwent splenic imaging with heat-denatured red blood cells labeled with technetium-99m (Tc-99m DRBC). The presenting problems included the heterotaxia syndrome, recurrent idiopathic thrombocytopenic purpura following splenectomy, mass in the left posterior hemithorax, and blunt abdominal trauma. In nine patients, the presence or absence of splenic tissue was established. A splenic hematoma was identified in the tenth patient. All patients were initially scanned with Tc-99m sulfur colloid (Tc-99m SC), and were selected for Tc-99m DRBC scintigraphy only after the results of the SC scans failed to establish the clinical problem beyond doubt. The availability of kits containing stannous ions, essential for efficient and stable labeling of red blood cells with Tc-99m and requiring only a small volume of blood, make splenic scintigraphy in children a relatively simple and definitive diagnostic procedure, when identification of splenic tissue is of clinical importance.

  16. Localization of lower gastrointestinal hemorrhage. Experience with red blood cells labeled in vitro with technetium Tc 99m

    International Nuclear Information System (INIS)

    Seventy-six patients clinically suspected of having lower gastrointestinal bleeding were studied by scintigraphy utilizing red blood cells labeled in vitro with technetium Tc 99m. Sixteen patients required emergency surgery; bleeding was accurately localized in 15 (94%). One patient (6%) had a normal scan. A 20-month mean follow-up of the 16 patients showed no recurrent bleeding. Of 60 patients not requiring emergency surgery, bleeding was localized in 11, but the bleeding ceased. Forty-nine of the 60 patients had normal scans and had no further hemorrhaging during hospitalization. A 21-month mean follow-up of 38 of the 49 patients showed no further bleeding episodes or surgical procedures in 29 patients; however, eight patients required surgical procedures, including seven for gastrointestinal malignancies. Scanning of red blood cells labeled in vitro with 99mTc is accurate and efficacious in localization of bleeding sites that require emergency surgery for lower gastrointestinal hemorrhage

  17. Pharmacokinetics on a microscale: visualizing Cy5-labeled oligonucleotide release from poly(n-butylcyanoacrylate nanocapsules in cells

    Directory of Open Access Journals (Sweden)

    Tomcin S

    2014-11-01

    Full Text Available Stephanie Tomcin,1 Grit Baier,1 Katharina Landfester,1 Volker Mailänder1,21Max Planck Institute for Polymer Research, 2University Medical Center of the Johannes Gutenberg University, III Medical Clinic, Mainz, GermanyAbstract: For successful design of a nanoparticulate drug delivery system, the fate of the carrier and cargo need to be followed. In this work, we fluorescently labeled poly(n-butylcyanoacrylate (PBCA nanocapsules as a shell and separately an oligonucleotide (20 mer as a payload. The nanocapsules were formed by interfacial anionic polymerization on aqueous droplets generated by an inverse miniemulsion process. After uptake, the PBCA capsules were shown to be round-shaped, endosomal structures and the payload was successfully released. Cy5-labeled oligonucleotides accumulated at the mitochondrial membrane due to a combination of the high mitochondrial membrane potential and the specific molecular structure of Cy5. The specificity of this accumulation at the mitochondria was shown as the uncoupler dinitrophenol rapidly diminished the accumulation of the Cy5-labeled oligonucleotide. Importantly, a fluorescence resonance energy transfer investigation showed that the dye-labeled cargo (Cy3/Cy5-labeled oligonucleotides reached its target site without degradation during escape from an endosomal compartment to the cytoplasm. The time course of accumulation of fluorescent signals at the mitochondria was determined by evaluating the colocalization of Cy5-labeled oligonucleotides and mitochondrial markers for up to 48 hours. As oligonucleotides are an ideal model system for small interfering RNA PBCA nanocapsules demonstrate to be a versatile delivery platform for small interfering RNA to treat a variety of diseases.Keywords: drug delivery, mitochondria, miniemulsion, colocalization

  18. Bright and stable CdSe/CdS@SiO2 nanoparticles suitable for long term cell labeling

    OpenAIRE

    Aubert, Tangi; Wassmuth, Daniel; Soenen, Stefaan; Braeckmans, Kevin; Hens, Zeger

    2014-01-01

    Semiconductor quantum dots (QDs) constitute very promising candidates as light emitters for numerous applications in the field of biotechnology, including cell labeling, in vivo imaging and diagnostics.[1] For such applications, semiconductor QDs represent an attractive alternative to classic organic fluorophores as they exhibit a higher brightness thanks to their large absorption cross-sections and high photoluminescence quantum yields. Nevertheless, QDs usually suffer from higly oxidative e...

  19. Inferior vena cava filter thrombus: A possible cause of an unanticipated finding of {sup 99m} Tc-labeled red blood cell scintigraphy

    Energy Technology Data Exchange (ETDEWEB)

    Song, Hee Sung; Choi, Joon Hyouk; Kim, Young Suk [Jeju National University School of Medicine, Jeju (Korea, Republic of)

    2016-06-15

    {sup 99m}Tc-labeled red blood cell scintigraphy, a sensitive and specific diagnostic test, is useful for patients suspected of suffering from active gastrointestinal bleeding. This study follows a case of a patient who was suspected of gastrointestinal bleeding after an inferior vena cava filter was inserted due to a deep vein thrombosis of the femoral vein. To evaluate an exact focus of bleeding, {sup 99m}Tc-labeled red blood cell scintigraphy was executed. Herein, an unanticipated finding of {sup 99m}Tc-labeled red blood cell scintigraphy probably due to a thrombus on the inferior vena cava filter is reported.

  20. Quantitative analysis of cell surface membrane proteins using membrane-impermeable chemical probe coupled with 18O labeling

    Science.gov (United States)

    Zhang, Haizhen; Brown, Roslyn N.; Qian, Wei-Jun; Monroe, Matthew E.; Purvine, Samuel O.; Moore, Ronald J.; Gritsenko, Marina A.; Shi, Liang; Romine, Margaret F; Fredrickson, James K.; Paša-Tolić, Ljiljana; Smith, Richard D.; Lipton, Mary S.

    2010-01-01

    We report a mass spectrometry-based strategy for quantitative analysis of cell surface membrane proteome changes. The strategy includes enrichment of surface membrane proteins using a membrane-impermeable chemical probe followed by stable isotope 18O labeling and LC-MS analysis. We applied this strategy for enriching membrane proteins expressed by Shewanella oneidensis MR-1, a gram-negative bacterium with known metal-reduction capability via extracellular electron transfer between outer membrane proteins and extracellular electron receptors. LC/MS/MS analysis resulted in the identification of about 400 proteins with 79% of them being predicted to be membrane localized. Quantitative aspects of the membrane enrichment were shown by peptide level 16O and 18O labeling of proteins from wild-type and mutant cells (generated from deletion of a type II secretion protein, GspD) prior to LC-MS analysis. Using a chemical probe labeled pure protein as an internal standard for normalization, the quantitative data revealed reduced abundances in ΔgspD mutant cells of many outer membrane proteins including the outer membrane c-cype cytochromes OmcA and MtrC, in agreement with previously investigation demonstrating that these proteins are substrates of the type II secretion system. PMID:20380418

  1. [Isolation and characterization of rapidly labelled high molecular RNA from freely suspended callus cells of parsley (Petroselinum sativum)].

    Science.gov (United States)

    Seitz, U; Richter, G

    1970-12-01

    By culturing of callus tissue originating from root explants of Petroselinum sativum in a synthetic liquid medium under aeration, freely suspended single cells and small clusters consisting of mostly five cells were obtained. The rapidly dividing cells did not exhibit any morphogenesis. Their nucleic acid metabolism was investigated by pulse experiments with (32)P-orthophosphate. Rapidly labelled RNA was prominently found associated with high molecular RNA. During the fractionation of the total nucleic acids on MAK columns it was eluted after the ribosomal RNA components. Its base ratio, however, differed from the latter in that the AMP content was higher than the GMP content. Sucrose gradient centrifugation and polyacrylamide gel electrophoresis resulted in the separation of the ribosomal RNA from the rapidly labelled RNA, thus proving the higher molecular weight of the latter. Based upon the migration in the gel a sedimentation coefficient of approximately 32S was calculated. The possible function of the heavy rapidly labelled RNA component as precursor of ribosomal RNA is discussed. PMID:24500301

  2. Separable Bilayer Microfiltration Device for Viable Label-free Enrichment of Circulating Tumour Cells

    Science.gov (United States)

    Zhou, Ming-Da; Hao, Sijie; Williams, Anthony J.; Harouaka, Ramdane A.; Schrand, Brett; Rawal, Siddarth; Ao, Zheng; Brennaman, Randall; Gilboa, Eli; Lu, Bo; Wang, Shuwen; Zhu, Jiyue; Datar, Ram; Cote, Richard; Tai, Yu-Chong; Zheng, Si-Yang

    2014-12-01

    The analysis of circulating tumour cells (CTCs) in cancer patients could provide important information for therapeutic management. Enrichment of viable CTCs could permit performance of functional analyses on CTCs to broaden understanding of metastatic disease. However, this has not been widely accomplished. Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture. Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably. Using multiple cancer cell lines spiked in healthy donor blood, the SB microfilter demonstrated high capture efficiency (78-83%), high retention of cell viability (71-74%), high tumour cell enrichment against leukocytes (1.7-2 × 103), and widespread ability to establish cultures post-capture (100% of cell lines tested). In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4-0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis. Our preliminary studies reflect the efficacy of the SB microfilter device to efficiently and reliably enrich viable CTCs in animal model studies, constituting an exciting technology for new insights in cancer research.

  3. Assessment of the effect of phytic acid on the labeling of blood cells and plasma proteins with Technetium-99m

    Energy Technology Data Exchange (ETDEWEB)

    Lima-Filho, Guilherme L.; Freitas, Rosimeire S.; Moreno, Silvana R.F.; Boasquevisque, Edson M.; Bernardo-Filho, Mario [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Inst. de Biologia. Dept. de Biofisica e Biometria]. E-mail: gllf@hotmail.com; Lima, Glaydes M.T. [Pernambuco Univ., Recife, PE (Brazil). Hospital das Clinicas; Catanho, Maria T.J.A. [Pernambuco Univ., Recife, PE (Brazil). Dept. de Biofisica e Radiobiologia

    2002-07-01

    Blood elements labeled with technetium-99m ({sup 99m} Tc) have been used in various procedures in nuclear medicine. We have investigated if phytic acid (PHY) could alter the labeling of blood elements with {sup 99m} Tc. Blood was incubated with different concentrations of PHY. Stannous chloride and {sup 99m}Tc, as sodium pertechnetate, were added. Blood was centrifuged and plasma (P) and blood cell (BC) were isolated. Samples of P and BC were also precipitated with trichloroacetic acid and centrifuged, and insoluble (IF) and soluble (SF) fractions were separated. The percentages of radioactivity (%ATI) in BC, IF-P and IF-BC were calculated. The %ATI decreased significantly (p < 0.05) in BC (95.08 {+-}1.94 to 80.68 {+-} 3.35), in IF-P (74.42 {+-}4.50 to 39.94{+-} 5.51) and in IF-BC (89.91{+-} 3.91 to 79.54 {+-} 5.42) in presence of PHY. These results suggest that the chelating property of PHY can modify the labeling of the BC, although other effects of PHY could be responsible. As PHY is found in many food and it could alter the labeling of blood elements with {sup 99m} Tc with possible undesirable effects, it is relevant to verify the necessity to repeat the examination and to evaluate the increase of the radiation dose to the patient. (author)

  4. Comparison of label-free and GFP multiphoton imaging of hair follicle-associated pluripotent (HAP) stem cells in mouse whiskers.

    Science.gov (United States)

    Uchugonova, Aisada; Cao, Wenluo; Hoffman, Robert M; Koenig, Karsten

    2015-01-01

    Hair-follicle-associated pluripotent (HAP) stem cells can differentiate into many cell types, including neurons and heart muscle cells, and have been shown to repair peripheral nerves and the spinal cord in mice. HAP stem cells can be obtained from each individual patient for regenerative medicine which overcomes problems with immune rejection. Previously, we have demonstrated that genetically-encoded protein markers such as GFP in transgenic mice can be used to visualize HAP stem cells in vivo by multiphoton tomography. Detection and visualization of stem cells in vivo without exogenous labels such as GFP would be important for human application. In the present report, we demonstrate label-free visualization of hair follicle stem cells in mouse whiskers by multiphoton tomography due to the intrinsic fluorophores such as NAD(P)H/flavins. We compared multiphoton tomography of GFP-labeled HAP stem cells and unlabeled stem cells in isolated mouse whiskers. We show that observation of HAP stem cells by label-free multiphoton tomography is comparable to detection using GFP-labeled stem cells. The results described here have important implications for detection and isolation of human HAP stem cells for regenerative medicine.

  5. Label-free and dynamic evaluation of cell-surface epidermal growth factor receptor expression via an electrochemiluminescence cytosensor.

    Science.gov (United States)

    Qiu, Youyi; Wen, Qingqing; Zhang, Lin; Yang, Peihui

    2016-04-01

    A label-free electrochemiluminescence (ECL) cytosensor was developed for dynamically evaluating of epidermal growth factor receptor (EGFR) expression on MCF-7 cancer cells based on the specific recognition of epidermal growth factor (EGF) with its receptor (EGFR). EGF-cytosensor was fabricated by in-situ electro-polymerization of polyaniline as substrate, using CdS quantum dots (CdS QDs) as ECL probe and gold nanoparticles (AuNPs) as a carrier for loading of EGF. AuNPs and CdS QDs were jointly attached on polyaniline surface to provide a sensitive and stable sensing interface, as well as a simple and label-free mode for ECL assay. Electron microscopy, atomic force microscopy (AFM) and electrochemical methods were employed to characterize the multilayer construction process of the sensing interface. The proposed EGF-cytosensor exhibited excellent analytical performance for MCF-7 cancer cells, ranging from 12 to 1.2 × 10(6) cells mL(-1), with a low detection limit of 12 cells mL(-1). Also, it was successfully applied in evaluating EGFR expression of cells surface, which was stimulated by some inhibitors or activator, and the results were confirmed by using flow cytometry and laser scanning confocal microscopy analysis. The proposed ECL cytosensor has potential applications in monitoring the dynamic variation of receptor molecules expression on cell surfaces in response to external stimulation by drugs and screening anti-cancer therapeutic agents. PMID:26838410

  6. Quantifying the length and variance of the eukaryotic cell cycle phases by a stochastic model and dual nucleoside pulse labelling.

    Directory of Open Access Journals (Sweden)

    Tom Serge Weber

    2014-07-01

    Full Text Available A fundamental property of cell populations is their growth rate as well as the time needed for cell division and its variance. The eukaryotic cell cycle progresses in an ordered sequence through the phases G1, S, G2, and M, and is regulated by environmental cues and by intracellular checkpoints. Reflecting this regulatory complexity, the length of each phase varies considerably in different kinds of cells but also among genetically and morphologically indistinguishable cells. This article addresses the question of how to describe and quantify the mean and variance of the cell cycle phase lengths. A phase-resolved cell cycle model is introduced assuming that phase completion times are distributed as delayed exponential functions, capturing the observations that each realization of a cycle phase is variable in length and requires a minimal time. In this model, the total cell cycle length is distributed as a delayed hypoexponential function that closely reproduces empirical distributions. Analytic solutions are derived for the proportions of cells in each cycle phase in a population growing under balanced growth and under specific non-stationary conditions. These solutions are then adapted to describe conventional cell cycle kinetic assays based on pulse labelling with nucleoside analogs. The model fits well to data obtained with two distinct proliferating cell lines labelled with a single bromodeoxiuridine pulse. However, whereas mean lengths are precisely estimated for all phases, the respective variances remain uncertain. To overcome this limitation, a redesigned experimental protocol is derived and validated in silico. The novelty is the timing of two consecutive pulses with distinct nucleosides that enables accurate and precise estimation of both the mean and the variance of the length of all phases. The proposed methodology to quantify the phase length distributions gives results potentially equivalent to those obtained with modern phase

  7. Rational design of 13C-labeling experiments for metabolic flux analysis in mammalian cells

    Directory of Open Access Journals (Sweden)

    Crown Scott B

    2012-05-01

    Full Text Available Abstract Background 13C-Metabolic flux analysis (13C-MFA is a standard technique to probe cellular metabolism and elucidate in vivo metabolic fluxes. 13C-Tracer selection is an important step in conducting 13C-MFA, however, current methods are restricted to trial-and-error approaches, which commonly focus on an arbitrary subset of the tracer design space. To systematically probe the complete tracer design space, especially for complex systems such as mammalian cells, there is a pressing need for new rational approaches to identify optimal tracers. Results Recently, we introduced a new framework for optimal 13C-tracer design based on elementary metabolite units (EMU decomposition, in which a measured metabolite is decomposed into a linear combination of so-called EMU basis vectors. In this contribution, we applied the EMU method to a realistic network model of mammalian metabolism with lactate as the measured metabolite. The method was used to select optimal tracers for two free fluxes in the system, the oxidative pentose phosphate pathway (oxPPP flux and anaplerosis by pyruvate carboxylase (PC. Our approach was based on sensitivity analysis of EMU basis vector coefficients with respect to free fluxes. Through efficient grouping of coefficient sensitivities, simple tracer selection rules were derived for high-resolution quantification of the fluxes in the mammalian network model. The approach resulted in a significant reduction of the number of possible tracers and the feasible tracers were evaluated using numerical simulations. Two optimal, novel tracers were identified that have not been previously considered for 13C-MFA of mammalian cells, specifically [2,3,4,5,6-13C]glucose for elucidating oxPPP flux and [3,4-13C]glucose for elucidating PC flux. We demonstrate that 13C-glutamine tracers perform poorly in this system in comparison to the optimal glucose tracers. Conclusions In this work, we have demonstrated that optimal tracer design does not

  8. Food Labels

    Science.gov (United States)

    ... How Can I Help a Friend Who Cuts? Food Labels KidsHealth > For Teens > Food Labels Print A ... have at least 95% organic ingredients. continue Making Food Labels Work for You The first step in ...

  9. Influence of Momordica charantia L. on the red and white blood cells labeling with 99mTc

    International Nuclear Information System (INIS)

    Full text: Momordica charantia L. is popularly known in Brazil as bitter melon and it's commonly used to treat several diseases as cancer, diabetes and to heal skin injuries. Many papers have been published showing the potential radio pharmacological activity of this plant due to its linkage with 99mTc through some protein fractions of the extract. In this study, it was evaluated the influence of Momordica charantia L extract , labeling ( in vitro) of blood elements with sodium pertechnetate (Na 99mTcO4). In the labeling of red blood cells (in vitro), blood samples were obtained from Wistar rats and incubated with different concentrations of M. charantia, for control group was used NaCl 0.9% and added stannous chloride (SnCl2) and 99mTc. The plasma fractions (P) and the cells (C) were separated and, also, precipitated with trichloroacetic acid at 5%, obtaining the soluble (SF) and insoluble (IF) fractions. The radioactivity rate (%ATl) of each fraction was calculated. The same methodology was applied for white blood cells but these cells were separated in advance by centrifugation at 1800 rpm during 15 minutes. There weren't alterations in the labeling of red blood cells in the concentrations tested of the extract when compared with the rate of the control group neither in the insoluble fractions. However, on the white blood cells it was noticed an increase in 99mTc uptake in the presence of M. charantia extract. So its possible to conclude, based on previous results obtained by our group, that the M. charantia L. could be used to evaluate inflammatory processes. (author)

  10. Mn-doped Zinc Sulphide nanocrystals for immunofluorescent labeling of epidermal growth factor receptors on cells and clinical tumor tissues

    International Nuclear Information System (INIS)

    The field of molecular detection and targeted imaging has evolved considerably with the introduction of fluorescent semiconductor nanocrystals. Manganese-doped zinc sulphide nanocrystals (ZnS:Mn NCs), which are widely used in electroluminescent displays, have been explored for the first time for direct immunofluorescent (IF) labeling of clinical tumor tissues. ZnS:Mn NCs developed through a facile wet chemistry route were capped using amino acid cysteine, conjugated to streptavidin and thereafter coupled to biotinylated epidermal growth factor receptor (EGFR) antibody utilizing the streptavidin–biotin linkage. The overall conjugation yielded stable EGFR antibody conjugated ZnS:Mn NCs (EGFR ZnS:Mn NCs) with a hydrodynamic diameter of 65 ± 15 nm, and having an intense orange–red fluorescence emission at 598 nm. Specific labeling of EGF receptors on EGFR+ve A431 cells in a co-culture with EGFR−ve NIH3T3 cells was demonstrated using these nanoprobes. The primary antibody conjugated fluorescent NCs could also clearly delineate EGFR over-expressing cells on clinical tumor tissues processed by formalin fixation as well as cryopreservation with a specificity of 86% and accuracy of 88%, in comparison to immunohistochemistry. Tumor tissues labeled with EGFR ZnS:Mn NCs showed good fluorescence emission when imaged after storage even at 15 months. Thus, ZnS nanobioconjugates with dopant-dependent and stable fluorescence emission show promise as an efficient, target-specific fluorophore that would enable long term IF labeling of any antigen of interest on clinical tissues. (paper)

  11. Mn-doped Zinc Sulphide nanocrystals for immunofluorescent labeling of epidermal growth factor receptors on cells and clinical tumor tissues

    Science.gov (United States)

    J, Aswathy; V, Seethalekshmy N.; R, Hiran K.; R, Bindhu M.; K, Manzoor; Nair, Shantikumar V.; Menon, Deepthy

    2014-11-01

    The field of molecular detection and targeted imaging has evolved considerably with the introduction of fluorescent semiconductor nanocrystals. Manganese-doped zinc sulphide nanocrystals (ZnS:Mn NCs), which are widely used in electroluminescent displays, have been explored for the first time for direct immunofluorescent (IF) labeling of clinical tumor tissues. ZnS:Mn NCs developed through a facile wet chemistry route were capped using amino acid cysteine, conjugated to streptavidin and thereafter coupled to biotinylated epidermal growth factor receptor (EGFR) antibody utilizing the streptavidin-biotin linkage. The overall conjugation yielded stable EGFR antibody conjugated ZnS:Mn NCs (EGFR ZnS:Mn NCs) with a hydrodynamic diameter of 65 ± 15 nm, and having an intense orange-red fluorescence emission at 598 nm. Specific labeling of EGF receptors on EGFR+ve A431 cells in a co-culture with EGFR-ve NIH3T3 cells was demonstrated using these nanoprobes. The primary antibody conjugated fluorescent NCs could also clearly delineate EGFR over-expressing cells on clinical tumor tissues processed by formalin fixation as well as cryopreservation with a specificity of 86% and accuracy of 88%, in comparison to immunohistochemistry. Tumor tissues labeled with EGFR ZnS:Mn NCs showed good fluorescence emission when imaged after storage even at 15 months. Thus, ZnS nanobioconjugates with dopant-dependent and stable fluorescence emission show promise as an efficient, target-specific fluorophore that would enable long term IF labeling of any antigen of interest on clinical tissues.

  12. Labeling of creatinine with technetium-99m

    Energy Technology Data Exchange (ETDEWEB)

    Yurt Lambrecht, F. [Ege Univ., Bornova, Izmir (Turkey). Dept. of Nuclear Applications, Inst. of Nuclear Sciences; Durkan, K. [Dokuz Eylul Univ., Buca, Izmir (Turkey). Chemistry Technicianship Program, Izmir Vocational School; Soylu, A. [Dokuz Eylul Univ., Narlidere, Izmir (Turkey). Dept. of Pediatrics, Medical Faculty

    2004-07-01

    Creatinine is a clinically important index of renal glomerular filtration rate. Urine creatinine levels can be used as a screening test to evaluate kidney function or can be part of the creatinine clearance test. In case of kidney dysfunction or muscle disorders the creatinine concentration in serum/plasma may rise to a higher value than in healthy body. Technetium- 99m has been used in nuclear medicine and in biomedical research to label molecular and cellular structures employed as radiotracers. {sup 99m}Tc is utilized to label molecules and cells, used as radiopharmaceuticals, and also to label biological species. It presents many desirable characteristics. SnCl{sub 2} method is frequently used as a reducing agent in the {sup 99m}Tc- labeling process. Creatinine metabolism might be investigated by using labeled {sup 99m}Tc- creatinine in healthy or uremic rats. (orig.)

  13. Stem Cell Research and Health Education

    Science.gov (United States)

    Eve, David J.; Marty, Phillip J.; McDermott, Robert J.; Klasko, Stephen K.; Sanberg, Paul R.

    2008-01-01

    Stem cells are being touted as the greatest discovery for the potential treatment of a myriad of diseases in the new millennium, but there is still much research to be done before it will be known whether they can live up to this description. There is also an ethical debate over the production of one of the most valuable types of stem cell: the…

  14. Internalization of cycloheptaamylose-dansyl chloride complex during labelling of surface membrane in living Paramecium aurelia cells.

    Science.gov (United States)

    Giordano, P A; Wyroba, E; Bottiroli, G

    1985-01-01

    Internalization of cycloheptaamylose-dansyl chloride complex during surface labelling of living long-term starved Paramecium aurelia cells has been observed. This process may be inhibited by pretreatment of the ciliates with dichloroisoproterenol. Uptake of cycloheptaamylose-dansyl chloride may be visualized only after UV preirradiation: the appearance of orange-fluorescing vacuoles of diameter 2.3-4.5 micron may then be observed. Microspectrographic analysis performed on the cells and dansyl derivatives indicates that this fluorescence is produced by a photochemical reaction of dansyl chloride - released from CDC complex inside the digestive vacuoles-under the influence of UV irradiation.

  15. Ultrasensitive detection of human liver hepatocellular carcinoma (HepG2) cells using a label-free aptasensor

    OpenAIRE

    Kashefi-Kheyrabadi, Leila; Mehrgardi, Masoud; Wiechec, Emilia; Anthony P. F. Turner; Tiwari, Ashutosh

    2014-01-01

    Liver cancer is one of the most common cancers in the world and has no effective cure, especially in later stages. The development of a tangible protocol for early diagnosis of this disease remains a major challenge. In the present manuscript, an aptamer-based, label-free electrochemical biosensor for the sensitive detection of HepG2, a hepatocellular carcinoma cell line, is described. The target cells are captured in a sandwich architecture using TLS11a aptamer covalently attached to a gold ...

  16. Use of enzyme label for quantitative evaluation of liposome adhesion on cell surface: studies with J774 macrophage monolayers.

    Science.gov (United States)

    Trubetskoy, V S; Dormeneva, E V; Tsibulsky, V P; Repin, V S; Torchilin, V P

    1988-07-01

    A method for quantitation of cell surface-bound liposomes utilizing J774 macrophage monolayers is developed. Surface-bound biotinyl-containing and 125I-labeled liposomes were quantified with avidin-peroxidase in an ELISA-like assay. Peroxidase substrate absorbance values were recalculated into the absolute amount of liposomal lipid using a special calibration plot. Total liposome uptake by macrophages was determined following the binding of 125I radioactivity. The approach suggested allows quantitative evaluation of the changes in the content of surface-adhered liposomes during their interaction with cells in vitro.

  17. Mass Spectrometric Analysis of the Cell Surface N-Glycoproteome by Combining Metabolic Labeling and Click Chemistry

    Science.gov (United States)

    Smeekens, Johanna M.; Chen, Weixuan; Wu, Ronghu

    2015-04-01

    Cell surface N-glycoproteins play extraordinarily important roles in cell-cell communication, cell-matrix interactions, and cellular response to environmental cues. Global analysis is exceptionally challenging because many N-glycoproteins are present at low abundances and effective separation is difficult to achieve. Here, we have developed a novel strategy integrating metabolic labeling, copper-free click chemistry, and mass spectrometry (MS)-based proteomics methods to analyze cell surface N-glycoproteins comprehensively and site-specifically. A sugar analog containing an azido group, N-azidoacetylgalactosamine, was fed to cells to label glycoproteins. Glycoproteins with the functional group on the cell surface were then bound to dibenzocyclooctyne-sulfo-biotin via copper-free click chemistry under physiological conditions. After protein extraction and digestion, glycopeptides with the biotin tag were enriched by NeutrAvidin conjugated beads. Enriched glycopeptides were deglycosylated with peptide- N-glycosidase F in heavy-oxygen water, and in the process of glycan removal, asparagine was converted to aspartic acid and tagged with 18O for MS analysis. With this strategy, 144 unique N-glycopeptides containing 152 N-glycosylation sites were identified in 110 proteins in HEK293T cells. As expected, 95% of identified glycoproteins were membrane proteins, which were highly enriched. Many sites were located on important receptors, transporters, and cluster of differentiation proteins. The experimental results demonstrated that the current method is very effective for the comprehensive and site-specific identification of the cell surface N-glycoproteome and can be extensively applied to other cell surface protein studies.

  18. Cellular penetration and nuclear importation properties of {sup 111}In-labeled and {sup 123}I-labeled HIV-1 tat peptide immunoconjugates in BT-474 human breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Cornelissen, Bart [Division of Nuclear Medicine, University Health Network, Toronto, ON, M5S 3M2 (Canada); Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); Hu, Meiduo [Division of Nuclear Medicine, University Health Network, Toronto, ON, M5S 3M2 (Canada); Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); McLarty, Kristin [Division of Nuclear Medicine, University Health Network, Toronto, ON, M5S 3M2 (Canada); Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); Costantini, Dan [Division of Nuclear Medicine, University Health Network, Toronto, ON, M5S 3M2 (Canada); Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); Reilly, Raymond M. [Division of Nuclear Medicine, University Health Network, Toronto, ON, M5S 3M2 (Canada) and Department of Medical Imaging, University of Toronto, Toronto, ON, M5S 3M2 (Canada) and Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada) and Toronto General Research Institute, University Health Network, Toronto, ON, M5S 3M2 (Canada)]. E-mail: raymond.reilly@utoronto.ca

    2007-01-15

    Introduction: Our objective was to compare the cell penetration and nuclear importation properties of {sup 111}In-labeled and {sup 123}I-labeled immunoconjugates (ICs) composed of 16-mer peptides (GRKKRRQRRRPPQGYG) derived from HIV-1 transactivator of transcription (tat) protein and anti-mouse IgG (mIgG) in BT-474 breast cancer (BC) cells. Methods: [{sup 111}In]tat ICs were constructed by site-specific conjugation of tat peptides to NaIO{sub 4} {sup -}-oxidized carbohydrates in the Fc domain of diethylenetriaminepentaacetic-acid-modified anti-mIgG antibodies. Immunoreactivity against mIgG was assessed in a competition assay. The kinetics of the accumulation of [{sup 111}In]anti-mIgG-tat IC and [{sup 123}I]anti-mIgG-tat ICs in BT-474 cells and the elimination of radioactivity from cells, cytoplasm or nuclei were determined. The effects of excess tat peptides or NH{sub 4}Cl (an inhibitor of endosomal acidification) on cellular uptake and nuclear importation of [{sup 111}In]anti-mIgG-tat were measured. Results: [{sup 111}In]anti-mIgG-tat was >97% radiochemically pure and exhibited preserved immunoreactivity with mIgG epitopes. [{sup 123}I]Anti-mIgG-tat penetrated BT-474 cells more rapidly than [{sup 111}In]anti-mIgG-tat ICs and achieved a 1.5-fold to a 2-fold higher uptake in cells and nuclei. Cell penetration and nuclear uptake of [{sup 111}In]anti-mIgG-tat were inhibited by excess tat peptides and NH{sub 4}Cl. Elimination of radioactivity from BT-474 cells and nuclei was more rapid and complete for {sup 123}I-labeled than for {sup 111}In-labeled anti-mIgG-tat ICs. Conclusion: Tat peptides derived from HIV-1 tat protein promoted the penetration and nuclear uptake of radioactivity following the incubation of {sup 111}In-labeled and {sup 123}I-labeled anti-mIgG antibodies with BT-474 human BC cells. {sup 111}In-labeled tat ICs are feasible for inserting radionuclides into cancer cells with potential for targeting intracellular and, particularly, nuclear epitopes for

  19. Nanoparticle Labeling of Bone Marrow-Derived Rat Mesenchymal Stem Cells: Their Use in Differentiation and Tracking

    Directory of Open Access Journals (Sweden)

    Ece Akhan

    2015-01-01

    Full Text Available Mesenchymal stem cells (MSCs are promising candidates for cellular therapies due to their ability to migrate to damaged tissue without inducing immune reaction. Many techniques have been developed to trace MSCs and their differentiation efficacy; however, all of these methods have limitations. Conjugated polymer based water-dispersible nanoparticles (CPN represent a new class of probes because they offer high brightness, improved photostability, high fluorescent quantum yield, and noncytotoxicity comparing to conventional dyes and quantum dots. We aimed to use this tool for tracing MSCs’ fate in vitro and in vivo. MSC marker expression, survival, and differentiation capacity were assessed upon CPN treatment. Our results showed that after CPN labeling, MSC markers did not change and significant number of cells were found to be viable as revealed by MTT. Fluorescent signals were retained for 3 weeks after they were differentiated into osteocytes, adipocytes, and chondrocytes in vitro. We also showed that the labeled MSCs migrated to the site of injury and retained their labels in an in vivo liver regeneration model. The utilization of nanoparticle could be a promising tool for the tracking of MSCs in vivo and in vitro and therefore can be a useful tool to understand differentiation and homing mechanisms of MSCs.

  20. Labeling HIV-1 virions with two fluorescent proteins allows identification of virions that have productively entered the target cell

    International Nuclear Information System (INIS)

    GFP-Vpr labeled HIV-1 virions have provided a method to visually examine the interactions between the virus and target cell during infection. However, existing methods to discriminate between virions that have been non-specifically endocytosed from those that have productively entered the host cell cytoplasm have remained problematic. Therefore, we examined the ability of a series of membrane-targeted fluorescent fusion protein constructs to be incorporated into virions. We find that a fluorescent protein fusion targeted to the plasma membrane by the addition of the N-terminal 15 amino acid sequence of c-Src (S15) is efficiently packaged into HIV virions. Using fluorescent proteins fused to this sequence, we have generated virions dually labeled with S15-mCherry and GFP-Vpr. Importantly, we can detect the loss of this S15-mCherry membrane signal following fusion. After infection with VSV-g pseudotyped HIV virions, we find a measurable, specific loss of membrane label during infection. This loss of fluorescence is not observed when fusion is prevented using bafilomycin A. This increased ability to discriminate between non-productively endocytosed virions and those actively undergoing steps in the infectious process will facilitate efforts to examine early steps in infection microscopically

  1. BrdU Pulse Labelling In Vivo to Characterise Cell Proliferation during Regeneration and Repair following Injury to the Airway Wall in Sheep

    Directory of Open Access Journals (Sweden)

    B. Yahaya

    2013-01-01

    Full Text Available The response of S-phase cells labelled with bromodeoxyuridine (BrdU in sheep airways undergoing repair in response to endobronchial brush biopsy was investigated in this study. Separate sites within the airway tree of anaesthetised sheep were biopsied at intervals prior to pulse labelling with BrdU, which was administered one hour prior to euthanasia. Both brushed and spatially disparate unbrushed (control sites were carefully mapped, dissected, and processed to facilitate histological analysis of BrdU labelling. Our study indicated that the number and location of BrdU-labelled cells varied according to the age of the repairing injury. There was little evidence of cell proliferation in either control airway tissues or airway tissues examined six hours after injury. However, by days 1 and 3, BrdU-labelled cells were increased in number in the airway wall, both at the damaged site and in the regions flanking either side of the injury. Thereafter, cell proliferative activity largely declined by day 7 after injury, when consistent evidence of remodelling in the airway wall could be appreciated. This study successfully demonstrated the effectiveness of in vivo pulse labelling in tracking cell proliferation during repair which has a potential value in exploring the therapeutic utility of stem cell approaches in relevant lung disease models.

  2. Poly (dopamine) coated superparamagnetic iron oxide nanocluster for noninvasive labeling, tracking, and targeted delivery of adipose tissue-derived stem cells

    Science.gov (United States)

    Liao, Naishun; Wu, Ming; Pan, Fan; Lin, Jiumao; Li, Zuanfang; Zhang, Da; Wang, Yingchao; Zheng, Youshi; Peng, Jun; Liu, Xiaolong; Liu, Jingfeng

    2016-01-01

    Tracking and monitoring of cells in vivo after transplantation can provide crucial information for stem cell therapy. Magnetic resonance imaging (MRI) combined with contrast agents is believed to be an effective and non-invasive technique for cell tracking in living bodies. However, commercial superparamagnetic iron oxide nanoparticles (SPIONs) applied to label cells suffer from shortages such as potential toxicity, low labeling efficiency, and low contrast enhancing. Herein, the adipose tissue-derived stem cells (ADSCs) were efficiently labeled with SPIONs coated with poly (dopamine) (SPIONs cluster@PDA), without affecting their viability, proliferation, apoptosis, surface marker expression, as well as their self-renew ability and multi-differentiation potential. The labeled cells transplanted into the mice through tail intravenous injection exhibited a negative enhancement of the MRI signal in the damaged liver-induced by carbon tetrachloride, and subsequently these homed ADSCs with SPIONs cluster@PDA labeling exhibited excellent repair effects to the damaged liver. Moreover, the enhanced target-homing to tissue of interest and repair effects of SPIONs cluster@PDA-labeled ADSCs could be achieved by use of external magnetic field in the excisional skin wound mice model. Therefore, we provide a facile, safe, noninvasive and sensitive method for external magnetic field targeted delivery and MRI based tracking of transplanted cells in vivo.

  3. On Orbit Immuno-Based, Label-Free, White Blood Cell Counting System with MicroElectroMechanical Sensor (MEMS) Technology (OILWBCS-MEMS) Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Aurora Flight Sciences Corporation and partner, Draper Laboratory, propose to develop an on-orbit immuno-based label-free white blood cell counting system using...

  4. On Orbit Immuno-Based, Label-Free, White Blood Cell Counting System with MicroElectroMechanical Sensor (MEMS) Technology (OILWBCS-MEMS) Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Aurora Flight Sciences Corporation and our partner, Draper Laboratory, propose to develop an on orbit immuno-based, label-free, white blood cell counting system for...

  5. Temporal Profiling and Pulsed SILAC Labeling Identify Novel Secreted Proteins during ex vivo Osteoblast Differentiation of Human Stromal Stem Cells

    DEFF Research Database (Denmark)

    Kristensen, Lars P; Chen, Li; Nielsen, Maria Overbeck;

    2012-01-01

    labeling to distinguish genuine secreted proteins from intracellular contaminants. We identified 466 potentially secreted proteins that were quantified at 5 time-points during 14-days ex vivo OB differentiation including 41 proteins known to be involved in OB functions. Among these, 315 proteins exhibited...... more than 2-fold up or down-regulation. The pulsed SILAC method revealed a strong correlation between the fraction of isotope labeling and the subset of proteins known to be secreted and involved in OB differentiation. We verified SILAC data using qRT-PCR analysis of 9 identified potential novel......It is well established that bone forming cells (osteoblasts) secrete proteins with autocrine, paracrine, and endocrine function. However, the identity and functional role for the majority of these secreted and differentially expressed proteins during the osteoblast (OB) differentiation process...

  6. Assessment of the effect of phytic acid on the labeling of blood cells and plasma proteins with Technetium-99m

    International Nuclear Information System (INIS)

    Blood elements labeled with technetium-99m (99m Tc) have been used in various procedures in nuclear medicine. We have investigated if phytic acid (PHY) could alter the labeling of blood elements with 99m Tc. Blood was incubated with different concentrations of PHY. Stannous chloride and 99mTc, as sodium pertechnetate, were added. Blood was centrifuged and plasma (P) and blood cell (BC) were isolated. Samples of P and BC were also precipitated with trichloroacetic acid and centrifuged, and insoluble (IF) and soluble (SF) fractions were separated. The percentages of radioactivity (%ATI) in BC, IF-P and IF-BC were calculated. The %ATI decreased significantly (p 99m Tc with possible undesirable effects, it is relevant to verify the necessity to repeat the examination and to evaluate the increase of the radiation dose to the patient. (author)

  7. Research on imaging, sensing, and characterization of cells at Research Center for Applied Sciences (RCAS), Academia Sinica

    Science.gov (United States)

    Tsai, Hui-Chen; Chang, Chun-Fang; Chen, Bi-Chang; Cheng, Ji-Yen; Chu, Chih-Wei; Han, Hsieh-Cheng; Hatanaka, Koji; Hsieh, Tung-Han; Lee, Chau-Hwang; Lin, Jung-Hsin; Tung, Yi-Chung; Wei, Pei-Kuen; Yang, Fu-Liang; Tsai, Din Ping

    2015-12-01

    Development of imaging, sensing, and characterization of cells at Research Center for Applied Sciences (RCAS) of Academia Sinica in Taiwan is progressing rapidly. The research on advanced lattice light sheet microscopy for temporal visualization of cells in three dimensions at sub-cellular resolution shows novel imaging results. Label-free observation on filopodial dynamics provides a convenient assay on cancer cell motility. The newly-developed software enables us to track the movement of two types of particles through different channels and reconstruct the co-localized tracks. Surface plasmon resonance (SPR) for detecting urinary microRNA for diagnosis of acute kidney injury demonstrates excellent sensitivity. A fully automated and integrated portable reader was constructed as a home-based surveillance system for post-operation hepatocellular carcinoma. New microfluidic cell culture devices for fast and accurate characterizations prove various diagnosis capabilities.

  8. Low molecular weight poly (2-dimethylamino ethylmethacrylate) polymers with controlled.positioned fluorescent labeling: Synthesis, characterization and in vitro interaction with human endothelial cells

    DEFF Research Database (Denmark)

    Flebus, Luca; Lombart, Francois; Sevrin, Chantal;

    2015-01-01

    ) fluorescent labeled PDMAEMA of low molecular weight (Mw) (below 15 kDa), controlling the position and density of fluorescein.The second goal was to analyze the possible difference in uptake and subcellular distribution of this labeled FF polycation between human umbilical vein endothelial cells (HUVEC) and h...... to a minor cytotoxicity compared to the higher ones.As main conclusion this study highlights the similitude in cell trafficking of FF PDMAEMA and data previously reported for PDMAEMA/DNA complexes....

  9. Manufacture of IRDye800CW-coupled Fe3O4 nanoparticles and their applications in cell labeling and in vivo imaging

    Directory of Open Access Journals (Sweden)

    Chen Zhongping

    2010-10-01

    Full Text Available Abstract Background In recent years, near-infrared fluorescence (NIRF-labeled iron nanoparticles have been synthesized and applied in a number of applications, including the labeling of human cells for monitoring the engraftment process, imaging tumors, sensoring the in vivo molecular environment surrounding nanoparticles and tracing their in vivo biodistribution. These studies demonstrate that NIRF-labeled iron nanoparticles provide an efficient probe for cell labeling. Furthermore, the in vivo imaging studies show excellent performance of the NIR fluorophores. However, there is a limited selection of NIRF-labeled iron nanoparticles with an optimal wavelength for imaging around 800 nm, where tissue autofluorescence is minimal. Therefore, it is necessary to develop additional alternative NIRF-labeled iron nanoparticles for application in this area. Results This study manufactured 12-nm DMSA-coated Fe3O4 nanoparticles labeled with a near-infrared fluorophore, IRDye800CW (excitation/emission, 774/789 nm, to investigate their applicability in cell labeling and in vivo imaging. The mouse macrophage RAW264.7 was labeled with IRDye800CW-labeled Fe3O4 nanoparticles at concentrations of 20, 30, 40, 50, 60, 80 and 100 μg/ml for 24 h. The results revealed that the cells were efficiently labeled by the nanoparticles, without any significant effect on cell viability. The nanoparticles were injected into the mouse via the tail vein, at dosages of 2 or 5 mg/kg body weight, and the mouse was discontinuously imaged for 24 h. The results demonstrated that the nanoparticles gradually accumulated in liver and kidney regions following injection, reaching maximum concentrations at 6 h post-injection, following which they were gradually removed from these regions. After tracing the nanoparticles throughout the body it was revealed that they mainly distributed in three organs, the liver, spleen and kidney. Real-time live-body imaging effectively reported the dynamic

  10. The labeling of C57BL/6j derived embryonic stem cells with enhanced green fluorescent protein

    Institute of Scientific and Technical Information of China (English)

    滕路; 张崇本; 尤洁芳; 尚克刚; 顾军

    2003-01-01

    Objective To labele MESPU35, a embryonic stem (ES) cell line derived from C57BL/6j mouse, with enhanced green fluorescent protein (EGFP) for further application.Methods The EGFP gene was controlled by the hybrid CA promoter/enhancer (CMV enhancer/ chicken beta-actin promoter/ beta-actin intron) to construct the vector of the transgene, pCA-EGFP. The vector was transfected into MESPU35 by electroporation.Results We generated EGFP expressing ES cells demonstrating normal properties. The green fluorescence of EGFP expressing cells was maintained in propagation of the ES cells for more than 30 passages as well as in differentiated cells. Cultured in suspension, the "green" ES cells aggregated, and formed embryoid bodies maintaining the green fluorescence at varying developmental stages. The "green" embryoid bodies could expand and differentiate into various types of cells, exhibiting ubiquitous green fluorescence. Conclusions The hybrid CA promoter/enhancer used to control the EGFP expressing ES cells, resulted in more intense and ubiquitous activity. The EGFP transfected cells yield bright green fluorescence, which can be visualized in real time and in situ. In addition, the ES cells, MESPU35, are derived from C57BL/6j mice, which are the most widely used in oncology, physiology and genetics. Compared to 129 substrains, C57BL/6j mice avoid a number of potential problems apparent in the other strains.

  11. Stem Cell Research: Unlocking the Mystery of Disease

    Science.gov (United States)

    ... Home Current Issue Past Issues From the Director: Stem Cell Research: Unlocking the Mystery of Disease Past Issues / Summer ... Zerhouni, NIH Director, described the need for expanding stem cell research. Recently, he spoke about stem cell research with ...

  12. Research progress of adult cardiac stem cells

    OpenAIRE

    Zheng, Nan; Ning-kun ZHANG; Lian-ru GAO

    2013-01-01

    The traditional view is that the heart is a terminal organ. This dogma, however, has been widely questioned with the discovery of adult cardiac stem cells (CSCs). Since CSCs have a highly self-renewal capacity and specific myocardial differentiation potential, nowadays they have been regarded as the most promising type of stem cells used in ischemic heart disease and other replacement therapy of end-stage heart disease. The present paper will focus on current results of scientific research on...

  13. The Cerebral Blood Flow Biomedical Informatics Research Network (CBFBIRN) database and analysis pipeline for arterial spin labeling MRI data.

    Science.gov (United States)

    Shin, David D; Ozyurt, I Burak; Liu, Thomas T

    2013-01-01

    Arterial spin labeling (ASL) is a magnetic resonance imaging technique that provides a non-invasive and quantitative measure of cerebral blood flow (CBF). After more than a decade of active research, ASL is now emerging as a robust and reliable CBF measurement technique with increased availability and ease of use. There is a growing number of research and clinical sites using ASL for neuroscience research and clinical care. In this paper, we present an online CBF Database and Analysis Pipeline, collectively called the Cerebral Blood Flow Biomedical Informatics Research Network (CBFBIRN) that allows researchers to upload and share ASL and clinical data. In addition to serving the role as a central data repository, the CBFBIRN provides a streamlined data processing infrastructure for CBF quantification and group analysis, which has the potential to accelerate the discovery of new scientific and clinical knowledge. All capabilities and features built into the CBFBIRN are accessed online using a web browser through a secure login. In this work, we begin with a general description of the CBFBIRN system data model and its architecture, then devote the remainder of the paper to the CBFBIRN capabilities. The latter part of our work is divided into two processing modules: (1) Data Upload and CBF Quantification Module; (2) Group Analysis Module that supports three types of analysis commonly used in neuroscience research. To date, the CBFBIRN hosts CBF maps and associated clinical data from more than 1,300 individual subjects. The data have been contributed by more than 20 different research studies, investigating the effect of various conditions on CBF including Alzheimer's, schizophrenia, bipolar disorder, depression, traumatic brain injury, HIV, caffeine usage, and methamphetamine abuse. Several example results, generated by the CBFBIRN processing modules, are presented. We conclude with the lessons learned during implementation and deployment of the CBFBIRN and our

  14. Preparation of liposome-coated oligonucleotide labeled with 99mTc and its uptake in vascular smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    To explore the preparation method of liposome-coated 99mTc-labeled antisense oligonucleotide (ASON),targeteing the proliferating cell nuclear antigen (PCNA), and to explore the biological characteristics and the uptake kinetics of a radiolabeled probe in vascular smooth muscle cells, an 18-base single-stranded antisense oligonucleotide targeting PCNA mRNA and the complementary strand (sense oligonucleotide, SON) were synthesized. The ASON (SON) was labeled with 99mTc, by conjugating the bifunctional chelator (hydrazino nicotinamide, HYNIC), and purified through a gel filtration column of Sephadex G-25. The product was then encapsulated in cationic liposome (oligofectamineTM). The radiolabeling efficiency, radiochemical purity, stability of the liposome-coated 99mTc-HYNIC-ASON in a phosphate buffered solution (PBS), and fresh human serum and its uptake rate were studied. There was no significant difference between the 99mTc radiolabeling efficiencies of HYNIC-ASON and HYNIC-SON, which were 60.04% ± 1.92% and 59.60% ± 2.53%, respectively (P > 0.05, n = 5). The radiochemical purity of the liposome-coated 99mTc-HYNIC-ASON was 94.70% ± 1.90% (n = 5). And after incubation with PBS and fresh human seAt 90 min after transfection, the uptake rate of the liposome-coated 99mTc-HYNIC-ASON reached its peak of 83.8% ±5.92% in vascular smooth muscle cells (VSMCs) and was much higher than that of the nonliposome-coated 99mTc-HYNIC-ASON, which was 11.16% ± 0.54% (P < 0.01, n = 4). The labeling method of PCNA ASON (SON) conjugated by HYNIC has been proved successful. The liposome was able to enhance the ASON (SON) uptake in VSMCs,and could be widely used as a safe, convenient, effective gene transfer carrier.

  15. Are reviewers obstructing stem cell research?

    Directory of Open Access Journals (Sweden)

    Bernard Binetruy

    2010-08-01

    Full Text Available Bernard BinetruyINSERM U626, Faculté de Médecine La Timone, Marseille, FranceA current controversy in stem cell research was published on the BBC website recently. Some stem cell researchers have said that "they believe a small group of scientists is effectively vetoing high quality science from publication in journals". They strongly suspected some reviewers to be deliberately sending back negative comments or asking for unnecessary experiments. Nature editor, Dr Philip Campbell, has said that "this idea is utterly false".

  16. An impedance-based cytotoxicity assay for real-time and label-free assessment of T-cell-mediated killing of adherent cells.

    Science.gov (United States)

    Peper, Janet Kerstin; Schuster, Heiko; Löffler, Markus W; Schmid-Horch, Barbara; Rammensee, Hans-Georg; Stevanović, Stefan

    2014-03-01

    The in vitro assessment of T-cell-mediated cytotoxicity plays an important and increasingly relevant role both in preclinical target evaluation and during immunomonitoring to accompany clinical trials employing targeted immunotherapies. For a long time, the gold standard for this purpose has been the chromium release assay (CRA). This end point assay, however, shows several disadvantages including the inevitable use of radioactivity. Based on electrical impedance measurements (using the xCELLigence system), we have established a label-free assay, facilitating the real-time monitoring of T-cell-mediated cytotoxicity. The coculture of peptide-specific T-cell lines with peptide-loaded target cells reproducibly led to a decrease in impedance due to induced apoptosis and detachment of target cells. Comparing our results to the standard CRA assay, we could demonstrate that impedance-based measurements show comparable results after short incubation periods (6h) but outperform the CRA both in reproducibility and sensitivity after prolonged incubation (24h), enabling the detection of target cell lysis with an effector to target ratio as low as 0.05:1. The impedance-based assay represents a valuable and highly sensitive tool for label-free real-time high throughput analysis of T-cell-mediated cytotoxicity.

  17. Effect of Peumus boldus on the labeling of red blood cells and plasma proteins with Technetium-99m

    Energy Technology Data Exchange (ETDEWEB)

    Wancke Reiniger, Ingrid; Fonseca de Oliveira, Joelma; Caldeira-de-Araujo, Adriano [Departamento de Biofisica e Biometria, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Bernardo-Filho, Mario [Instituto Nacional de Cancer, Centro de Pesquisa Basica, Rio de Janeiro, RJ (Brazil)

    1999-08-01

    Peumus boldus is used in popular medicine in Brazil. The influence of Peumus boldus on the labeling of red blood cells and plasma proteins with {sup 99m}Tc was studied. Stannous chloride and {sup 99m}Tc pertechnetate were incubated with blood and a tincture of Peumus boldus. Aliquots of plasma and blood cells were isolated from the mixture and treated with trichloroacetic acid (TCA). After separation, analysis of the soluble and insoluble fractions showed a rapid uptake of the radioactivity by blood cells in the presence of the drug, whereas there was a slight decrease in the amount of {sup 99m}Tc radioactivity in the TCA-insoluble fraction of plasma.

  18. Effect of Peumus boldus on the labeling of red blood cells and plasma proteins with technetium-99m.

    Science.gov (United States)

    Reiniger, I W; de Oliveira, J F; Caldeira-de-Araújo, A; Bernardo-Filho, M

    1999-08-01

    Peumus boldus is used in popular medicine in Brazil. The influence of Peumus boldus on the labeling of red blood cells and plasma proteins with 99mTc was studied. Stannous chloride and 99mTc pertechnetate were incubated with blood and a tincture of Peumus boldus. Aliquots of plasma and blood cells were isolated from the mixture and treated with trichloroacetic acid (TCA). After separation, analysis of the soluble and insoluble fractions showed a rapid uptake of the radioactivity by blood cells in the presence of the drug, whereas there was a slight decrease in the amount of 99mTc radioactivity in the TCA-insoluble fraction of plasma. PMID:10376326

  19. Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Applied to Quantitative Proteomics of Bacillus subtilis

    DEFF Research Database (Denmark)

    Soufi, Boumediene; Kumar, C.; Gnad, F.;

    2010-01-01

    We applied stable isotope labeling by amino acids in cell culture (SILAC) to large-scale quantitative proteomics analyses of the model bacterium Bacillus subtilis in two physiological conditions: growth on succinate and growth under phosphate starvation. Using a B. subtilis strain auxotrophic...... of the most comprehensive quantitative proteomics studies in bacteria, covering more than 75% of the B. subtilis genes expressed in the log phase of growth. Furthermore, we detect and quantify dynamics of 35 Ser/Thr/Tyr phosphorylation sites under growth on succinate, and 10 phosphorylation sites under...

  20. Ibrutinib versus temsirolimus in patients with relapsed or refractory mantle-cell lymphoma: an international, randomised, open-label, phase study

    OpenAIRE

    Dreyling, Martin; Jurczak, Wojciech; Jerkeman, Mats; Silva, Rodrigo Santucci; Rusconi, Chiara; Trneny, Marek; Offner, Fritz; Caballero, Dolores; Joao, Cristina; Witzens-Harig, Mathias; Hess, Georg; Bence-Bruckler, Isabelle; Cho, Seok-Goo; Bothos, John; Goldberg, Jenna D.

    2016-01-01

    Background: Mantle-cell lymphoma is an aggressive B-cell lymphoma with a poor prognosis. Both ibrutinib and temsirolimus have shown single-agent activity in patients with relapsed or refractory mantle-cell lymphoma. We undertook a phase 3 study to assess the efficacy and safety of ibrutinib versus temsirolimus in relapsed or refractory mantle-cell lymphoma. Methods: This randomised, open-label, multicentre, phase 3 clinical trial enrolled patients with relapsed or refractory mantle-cell l...

  1. Intercellular imaging by a polyarginine derived cell penetrating peptide labeled magnetic resonance contrast agent,diethylenetriamine pentaacetic acid gadolinium

    Institute of Scientific and Technical Information of China (English)

    GUO You-min; LIU Min; YANG Jun-le; GUO Xiao-juan; WANG Si-cen; DUAN Xiao-yi; WANG Peng

    2007-01-01

    Background The cellular plasma membrane represents a natural barrier to many exogenous molecules including magnetic resonance (MR) contrast agent. Cell penetrating peptide (CPP) is used to internalize proteins, peptides, and radionuclide. This study was undertaken to assess the value of a new intracellular MR contrast medium, CPP labeled diethylenetriamine pentaacetic acid gadolinium (Gd-DTPA) in molecular imaging in vitro. Methods Fluorescein-5-isothiocyanate (FITC) and Gd-DTPA respectively labeled with CPP (FITC-CPP, Gd-DTPA-CPP) were synthesized by the solid-phase method. Human hepatic cancer cell line-HepG2 was respectively stained by FITC-CPP and FITC to observe the uptake and intracellular distribution. HepG2 was respectively incubated with 100 nmol/ml Gd-DTPA-CPP for 0, 10, 30, 60 minutes, and imaged by MR for studying the relationship between the incubation time and T1WI signal. The cytotoxicity to NIH3T3 fibroblasts cells was measured by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide reduction assay (MTT). Results The molecular weights of CPP labeled imaging agents, which were determined by MALDI mass spectrometry (FITC-CPP MW=2163.34, Gd-DTPA-CPP MW=2285.99), were similar to the calculated molecular weights. Confocal microscopy suggested HepG2 translocated FITC-CPP in cytoplasm and nucleus independent with the incubation temperature. MR images showed HepG2 uptaken Gd-DTPA-CPP had a higher T1 weighted imaging (T1WI) signal, and that the T1WI signal intensity was increasing in a time-dependent manner (r=0.972, P=0.001), while the signal intensity between the cells incubated by Gd-DTPA for 60 minutes and the controlled cells was not significantly different (P=0.225). By MTT, all concentrations from 50 nmol/ml to 200 nmol/ml had no significant (F=0.006, P=1.000) effect on cell viability of mouse NIH3T3 fibroblasts, compared with the control group. Conclusions The newly constructed CPP based on polyarginine can translocate cells by carrying FITC

  2. Whole cell, label free protein quantitation with data independent acquisition: quantitation at the MS2 level.

    Science.gov (United States)

    McQueen, Peter; Spicer, Vic; Schellenberg, John; Krokhin, Oleg; Sparling, Richard; Levin, David; Wilkins, John A

    2015-01-01

    Label free quantitation by measurement of peptide fragment signal intensity (MS2 quantitation) is a technique that has seen limited use due to the stochastic nature of data dependent acquisition (DDA). However, data independent acquisition has the potential to make large scale MS2 quantitation a more viable technique. In this study we used an implementation of data independent acquisition--SWATH--to perform label free protein quantitation in a model bacterium Clostridium stercorarium. Four tryptic digests analyzed by SWATH were probed by an ion library containing information on peptide mass and retention time obtained from DDA experiments. Application of this ion library to SWATH data quantified 1030 proteins with at least two peptides quantified (∼ 40% of predicted proteins in the C. stercorarium genome) in each replicate. Quantitative results obtained were very consistent between biological replicates (R(2) ∼ 0.960). Protein quantitation by summation of peptide fragment signal intensities was also highly consistent between biological replicates (R(2) ∼ 0.930), indicating that this approach may have increased viability compared to recent applications in label free protein quantitation. SWATH based quantitation was able to consistently detect differences in relative protein quantity and it provided coverage for a number of proteins that were missed in some samples by DDA analysis. PMID:25348682

  3. Insect cell culture in research: Indian scenario.

    Science.gov (United States)

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

    2005-06-01

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

  4. Commercial speech and off-label drug uses: what role for wide acceptance, general recognition and research incentives?

    Science.gov (United States)

    Gilhooley, Margaret

    2011-01-01

    approval. Distributions of information about unapproved uses should not be acceptable unless experts consider the expanded use to be generally recognized as safe and effective based on adequate studies. The last part of this paper considers the need to develop better research incentives to encourage more testing and post-market risk surveillance by drug makers on off-label uses of their drugs. Violations of the Federal Food Drug and Cosmetic Act (FFDCA) can be considered violations of the False Claims Act, which opens the way to fraud and abuse suits. The scale of penalties involved in these suits may lead to more examination of the scope of FDA regulation and commercial speech protections. Thus this symposium's consideration of these issues is timely and important. PMID:21847881

  5. The Cerebral Blood Flow Biomedical Informatics Research Network (CBFBIRN Database and Analysis Pipeline for Arterial Spin Labeling MRI Data

    Directory of Open Access Journals (Sweden)

    David D. Shin

    2013-10-01

    Full Text Available Arterial spin labeling (ASL is a MRI technique that provides a noninvasive and quantitative measure of cerebral blood flow (CBF. After more than a decade of active research, ASL is now emerging as a robust and reliable CBF measurement technique with increased availability and ease of use. There is a growing number of research and clinical sites using ASL for neuroscience research and clinical care. In this paper, we present an online CBF Database and Analysis Pipeline, collectively called the CBFBIRN that allows researchers to upload and share ASL and clinical data. In addition to serving the role as a central data repository, the CBFBIRN provides a streamlined data processing infrastructure for CBF quantification and group analysis, which has the potential to accelerate the discovery of new scientific and clinical knowledge. All capabilities and features built into the CBFBIRN are accessed online using a web browser through a secure login. In this work, we begin with a general description of the CBFBIRN system data model and its architecture, then devote the remainder of the paper to the CBFBIRN capabilities. The latter part of our work is divided into two processing modules: 1 Data Upload and CBF Quantification Module; 2 Group Analysis Module that supports three types of analysis commonly used in neuroscience research. To date, the CBFBIRN hosts CBF maps and associated clinical data from more than 1300 individual subjects. The data have been contributed by more than 20 different research studies, investigating the effect of various conditions on CBF including Alzheimer’s, schizophrenia, bipolar disorder, depression, traumatic brain injury, HIV, caffeine usage and Methamphetamine abuse. Several example results, generated by the CBFBIRN processing modules, are presented. We conclude with the lessons learned during implementation and deployment of the CBFBIRN and our experience in promoting data sharing.

  6. CellTracker Green labelling vs. rose bengal staining: CTG wins by points in distinguishing living from dead anoxia-impacted copepods and nematodes

    Directory of Open Access Journals (Sweden)

    M. Grego

    2013-07-01

    Full Text Available Hypoxia and anoxia have become a key threat to shallow coastal seas. Much is known about their impact on macrofauna, less on meiofauna. In an attempt to shed more light on the latter group, in particular from a process-oriented view, we experimentally induced short-term anoxia (1 week in the northern Adriatic Sea (Mediterranean and examined the two most abundant meiofauna taxa – harpacticoid copepods and nematodes. Both taxa also represent different ends of the tolerance spectrum, with copepods being the most sensitive and nematodes among the most tolerant. We compared two methods: CellTracker Green (CTG – new labelling approach for meiofauna – with the traditional rose bengal (RB staining method. CTG binds to active enzymes and therefore colours live organisms only. The two methods show considerable differences in the number of living and dead individuals of both meiofauna taxa. Generally, RB will stain dead but not yet decomposed copepods and nematodes equally as it does live ones. Specifically, RB significantly overestimated the number of living copepods in all sediment layers in anoxic samples, but not in any normoxic samples. In contrast, for nematodes, the methods did not show such a clear difference between anoxia and normoxia. RB overestimated the number of living nematodes in the top sediment layer of normoxic samples, which implies an overestimation of the overall live nematofauna. For monitoring and biodiversity studies, the RB method might be sufficient, but for more precise quantification of community degradation, especially after an oxygen depletion event, CTG labelling is a better tool. Moreover, it clearly highlights the surviving species within the copepod or nematode community. As already accepted for foraminiferal research, we demonstrate that the CTG labelling is also valid for other meiofauna groups.

  7. Low molecular weight alkyl-polycation wrapped magnetite nanoparticle clusters as MRI probes for stem cell labeling and in vivo imaging.

    Science.gov (United States)

    Liu, Gang; Wang, Zhiyong; Lu, Jian; Xia, Chunchao; Gao, Fabao; Gong, Qiyong; Song, Bin; Zhao, Xuna; Shuai, Xintao; Chen, Xiaoyuan; Ai, Hua; Gu, Zhongwei

    2011-01-01

    Superparamagnetic iron oxide (SPIO) nanoparticles are potential probes for noninvasive cell tracking, but the design of safe probes coupled with high labeling efficiency is still an important objective for such application. In this study, an efficient SPIO probe has been developed for mesenchymal stem cells (MSCs) labeling and tracking. Different from many other systems involving high molecular polycations, we chose low molecular weight amphiphilic PEI2k to form stable nanocomplexes with SPIO nanoparticles. The probe can hold multiple SPIO nanoparticles with a controlled clustering structure, leading to much higher T(2) relaxivities compared to single SPIO nanoparticles. Labeled MSCs are unaffected in their viability, proliferation, or differentiation capacity. The iron uptake process in MSCs displays a time- and dose-dependent behavior. Transmission electron microscopy reveals that the nanoprobes are internalized into the cytoplasm of MSCs. Subcutaneous injection of the labeled MSCs dispersed in a collagen type I hydrogel showed strong image contrast against unlabeled cells under a clinical 3T magnetic resonance imaging (MRI) scanner up to 19 days post-transplantation. This study provides an important alternative to label MSCs at optimized low dosages with high efficiency, and the probe may be useful to label other biologically important cells for imaging studies.

  8. Skeletal stem cell isolation: A review on the state-of-the-art microfluidic label-free sorting techniques.

    Science.gov (United States)

    Xavier, Miguel; Oreffo, Richard O C; Morgan, Hywel

    2016-01-01

    Skeletal stem cells (SSC) are a sub-population of bone marrow stromal cells that reside in postnatal bone marrow with osteogenic, chondrogenic and adipogenic differentiation potential. SSCs reside only in the bone marrow and have organisational and regulatory functions in the bone marrow microenvironment and give rise to the haematopoiesis-supportive stroma. Their differentiation capacity is restricted to skeletal lineages and therefore the term SSC should be clearly distinguished from mesenchymal stem cells which are reported to exist in extra-skeletal tissues and, critically, do not contribute to skeletal development. SSCs are responsible for the unique regeneration capacity of bone and offer unlimited potential for application in bone regenerative therapies. A current unmet challenge is the isolation of homogeneous populations of SSCs, in vitro, with homogeneous regeneration and differentiation capacities. Challenges that limit SSC isolation include a) the scarcity of SSCs in bone marrow aspirates, estimated at between 1 in 10-100,000 mononuclear cells; b) the absence of specific markers and thus the phenotypic ambiguity of the SSC and c) the complexity of bone marrow tissue. Microfluidics provides innovative approaches for cell separation based on bio-physical features of single cells. Here we review the physical principles underlying label-free microfluidic sorting techniques and review their capacity for stem cell selection/sorting from complex (heterogeneous) samples. PMID:27236022

  9. Imaging of VSOP labeled stem cells in agarose phantoms with susceptibility weighted and T2* weighted MR Imaging at 3T: determination of the detection limit.

    Directory of Open Access Journals (Sweden)

    Donald Lobsien

    Full Text Available OBJECTIVES: This study aimed to evaluate the detectability of stem cells labeled with very small iron oxide particles (VSOP at 3T with susceptibility weighted (SWI and T2* weighted imaging as a methodological basis for subsequent examinations in a large animal stroke model (sheep. MATERIALS AND METHODS: We examined ovine mesenchymal stem cells labeled with VSOP in agarose layer phantoms. The experiments were performed in 2 different groups, with quantities of 0-100,000 labeled cells per layer. 15 different SWI- and T2*-weighted sequences and 3 RF coils were used. All measurements were carried out on a clinical 3T MRI. Images of Group A were analyzed by four radiologists blinded for the number of cells, and rated for detectability according to a four-step scale. Images of Group B were subject to a ROI-based analysis of signal intensities. Signal deviations of more than the 0.95 confidence interval in cell containing layers as compared to the mean of the signal intensity of non cell bearing layers were considered significant. RESULTS: GROUP A: 500 or more labeled cells were judged as confidently visible when examined with a SWI-sequence with 0.15 mm slice thickness. Group B: 500 or more labeled cells showed a significant signal reduction in SWI sequences with a slice thickness of 0.25 mm. Slice thickness and cell number per layer had a significant influence on the amount of detected signal reduction. CONCLUSION: 500 VSOP labeled stem cells could be detected with SWI imaging at 3 Tesla using an experimental design suitable for large animal models.

  10. What Undergraduates Misunderstand about Stem Cell Research

    Science.gov (United States)

    Halverson, Kristy Lynn; Freyermuth, Sharyn K.; Siegel, Marcelle A.; Clark, Catharine G.

    2010-01-01

    As biotechnology-related scientific advances, such as stem cell research (SCR), are increasingly permeating the popular media, it has become ever more important to understand students' ideas about this issue. Very few studies have investigated learners' ideas about biotechnology. Our study was designed to understand the types of alternative…

  11. Medical Research on Stem Cells to Continue

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    China will maintain its opposition to human reproductive cloning but will continue to allow closely monitored embryo stem cell research for the treatment and prevention of disease, said Wang Hongguang, president of the China National Center for Biotechnology Development, on February 20 in Beijing.

  12. Web Resources for Stem Cell Research

    Institute of Scientific and Technical Information of China (English)

    Ting Wei; Xing Peng; Lili Ye; Jiajia Wang; Fuhai Song; Zhouxian Bai; Guangchun Han; Fengmin Ji; Hongxing Lei

    2015-01-01

    In this short review, we have presented a brief overview on major web resources relevant to stem cell research. To facilitate more efficient use of these resources, we have provided a pre-liminary rating based on our own user experience of the overall quality for each resource. We plan to update the information on an annual basis.

  13. Morphologic alterations on red blood cells labeled with technetium-99m: the effect of Mentha crispa L. (hortela) extract

    International Nuclear Information System (INIS)

    The use of natural products, as medicinal plants, is very frequent in the world. Mentha crispa L. (M. crispa) is utilized in herbal medicine. Blood elements labeled with technetium-99m (99mTc) are used in nuclear medicine procedures and this labeling process may be altered by drugs. We have investigated the possibility of M. crispa extract being capable to alter the labeling of blood elements with 99mTc. Blood was incubated with M. crispa extract in various concentrations (6.25, 12.5, 25, 50 and 100%). Stannous chloride solution and Tc-99m, as sodium pertechnetate, were added. Blood was centrifuged and plasma (P) and blood cells (BC) were isolated. Samples of P and BC were also precipitated, centrifuged and insoluble (IF) and soluble (SF) separated. The percentage of radioactivity (%ATI) in BC, IF-P and IF-BC was calculated. Histological evaluations of the red blood cells (RBC) were performed with blood samples treated with various concentrations of M. Crispa L. and the morphology of the RBC was observed under optical microscope. Important morphological alterations expressed by mean of the perimeter/area of the RBC treated with M. crispa: 6.25% (0.67 ± 0.02), 12.5% (0.77 ± 0.03), 25% (0.73 ± 0.04), 50% (0.76 ± 0.04), 100% (0.69 ± 0.08) and the control cells (0.67 ± 0.05). The %ATI decreased: (i) on BC from 97.3 ± 1.92 to 60.0 ± 2.44; (ii) on IF-P from 74.8 ± 3.78 to 9.99 ± 3.61; (iii) on IF-BC from 88.6 ± 5.41 to 58.4 ± 11.55. The perimeter/area of the RBC showed significant differences (P>0.01) when compared 6.25% and 12.5%, and when compared 6.25% and 50% of M. Crispa L. extract. These findings could also justify the decrease of the labeling of BC with 99mTc in presence of M. Crispa extract

  14. Research progress of adult cardiac stem cells

    Directory of Open Access Journals (Sweden)

    Nan ZHENG

    2013-04-01

    Full Text Available The traditional view is that the heart is a terminal organ. This dogma, however, has been widely questioned with the discovery of adult cardiac stem cells (CSCs. Since CSCs have a highly self-renewal capacity and specific myocardial differentiation potential, nowadays they have been regarded as the most promising type of stem cells used in ischemic heart disease and other replacement therapy of end-stage heart disease. The present paper will focus on current results of scientific research on human adult CSCs and epicardium-derived cell (EPDC, as well as the treatment strategies in the field of cardiac regeneration, and the problems and prospect disclosed in the research.

  15. Proliferation kinetics of early hemopoietic precursor cells with self sustaining capacity in the mouse, studied with 125-I-labeled iodo-deoxyuridine

    International Nuclear Information System (INIS)

    With a new labelling technique in radiation chimeras, an attempt was made to determine the duration of the phases of the stem cell cycle including shortest and mean generation time and to estimate the number of hemopoietic stem cells per unit of bone marrow cellularity. The DNA of bone marrow cells in DNA synthesis was labeled with 5-125I-2'- deoxyuridine. The labeled cells were followed after being transfused into fatally irradiated mice. The stem cells were found to have a half-time of about 4.3 days in the donor mice. The average time in the population, i.e. the turnover time of the stem cells, was 6.2 days. The half-time did not change significantly even after transfusion of bone marrow into lethally irradiated recipient mice. Tritiated thymidine (3H-TdR) suicide technique revealed that bone marrow stem cells seeding to the spleens and to the femurs of lethally irradiated recipients behaved differently - S-phase in cells seeding to femurs being shorter. The radiosensitivity of stem cells in S-phase had a D0 of 80 rad whereas stem cells distributed throughout the whole cell cycle had a D0 of 185 rad. The respective extrapolation numbers were 1.23 and 1.14. It is calculated that 2-7% of all nucleated bone marrow cells belong to self renewing stem cell populations. The method described provides a new approach for the study of hematological stem cells. (author)

  16. 3 CFR - Guidelines for Human Stem Cell Research

    Science.gov (United States)

    2010-01-01

    ..., scientifically worthy human stem cell research, including human embryonic stem cell research, to the extent... Guidelines apply to the expenditure of NIH funds for research using human embryonic stem cells and certain... responsible research with human embryonic stem cells has the......

  17. In vitro detection of mdr1 mRNA in murine leukemia cells with {sup 111}In-labeled oligonucleotide

    Energy Technology Data Exchange (ETDEWEB)

    Bai Jingming; Yokoyama, Kunihiko; Kinuya, Seigo; Michigishi, Takatoshi; Tonami, Norihisa [Kanazawa University Graduate School of Medical Sciences, Department of Biotracer Medicine (Nuclear Medicine), Kanazawa (Japan); Shiba, Kazuhiro [Kanazawa University, Radioisotope Center, Kanazawa (Japan); Matsushita, Ryo [Kanazawa University, Laboratory for Development of Medicine, Faculty of Pharmaceutical Sciences, Kanazawa (Japan); Nomura, Masaaki [Kanazawa University Hospital, Hospital Pharmacy, Kanazawa (Japan)

    2004-11-01

    The feasibility of intracellular mdr1 mRNA expression detection with radiolabeled antisense oligonucleotide (ODN) was investigated in the murine leukemia cell line, P388/S, and its subclonal, adriamycin-resistant cell line, P388/R. The expression level of mdr1 mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Existence of the multidrug resistance (MDR) phenomenon was assessed via cellular uptake of {sup 99m}Tc-sestamibi (MIBI), a known substrate for P-glycoprotein. A 15-mer phosphorothioate antisense ODN complementary to the sequences located at -1 to 14 of mdr1 mRNA and its corresponding sense ODN were conjugated with the cyclic anhydride of diethylene triamine penta-acetic acid (cDTPA) via an amino group linked to the terminal phosphate at the 5' end at pH 8-9. The DTPA-ODN complexes at concentrations of 0.1-17.4 {mu}Mwere reacted with {sup 111}InCl{sub 3} at pH 5 for 1 h. The hybridization affinity of labeled ODN was evaluated with size-exclusion high-performance liquid chromatography following incubation with the complementary sequence. Cellular uptake of labeled ODN was examined in vitro. Furthermore, enhancing effects of synthetic lipid carriers (Transfast) on transmembrane delivery of ODN were assessed. P388/R cells displayed intense mdr1 mRNA expression in comparison with P388/S cells. {sup 99m}Tc-MIBI uptake in P388/S cells was higher than that in P388/R cells. Specific radioactivity up to 1,634 MBq/nmol was achieved via elevation of added radioactivity relative to ODN molar amount. The hybridization affinity of antisense {sup 111}In-ODN was preserved at approximately 85% irrespective of specific activity. Cellular uptake of antisense {sup 111}In-ODN did not differ from that of sense {sup 111}In-ODN in either P388/S cells or P388/R cells. However, lipid carrier incorporation significantly increased transmembrane delivery of {sup 111}In-ODN; moreover, specific uptake of antisense {sup 111}In-ODN was demonstrated in P388/R

  18. Cell Science and Cell Biology Research at MSFC: Summary

    Science.gov (United States)

    2003-01-01

    The common theme of these research programs is that they investigate regulation of gene expression in cells, and ultimately gene expression is controlled by the macromolecular interactions between regulatory proteins and DNA. The NASA Critical Path Roadmap identifies Muscle Alterations and Atrophy and Radiation Effects as Very Serious Risks and Severe Risks, respectively, in long term space flights. The specific problem addressed by Dr. Young's research ("Skeletal Muscle Atrophy and Muscle Cell Signaling") is that skeletal muscle loss in space cannot be prevented by vigorous exercise. Aerobic skeletal muscles (i.e., red muscles) undergo the most extensive atrophy during long-term space flight. Of the many different potential avenues for preventing muscle atrophy, Dr. Young has chosen to study the beta-adrenergic receptor (betaAR) pathway. The reason for this choice is that a family of compounds called betaAR agonists will preferentially cause an increase in muscle mass of aerobic muscles (i.e., red muscle) in animals, potentially providing a specific pharmacological solution to muscle loss in microgravity. In addition, muscle atrophy is a widespread medical problem in neuromuscular diseases, spinal cord injury, lack of exercise, aging, and any disease requiring prolonged bedridden status. Skeletal muscle cells in cell culture are utilized as a model system to study this problem. Dr. Richmond's research ("Radiation & Cancer Biology of Mammary Cells in Culture") is directed toward developing a laboratory model for use in risk assessment of cancer caused by space radiation. This research is unique because a human model will be developed utilizing human mammary cells that are highly susceptible to tumor development. This approach is preferential over using animal cells because of problems in comparing radiation-induced cancers between humans and animals.

  19. Quantitative Proteomic Analysis of Mouse Embryonic Fibroblasts and Induced Pluripotent Stem Cells Using 16O /18O labeling

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Xin; Tian, Changhai; Liu, Miao; Wang, Yongxiang; Tolmachev, Aleksey V.; Sharma, Seema; Yu, Fang; Fu, Kai; Zheng, Jialin; Ding, Shi-Jian

    2012-04-06

    Induced pluripotent stem cells (iPSC) hold great promise for regenerative medicine as well as for investigations into the pathogenesis and treatment of various diseases. Understanding of key intracellular signaling pathways and protein targets that control development of iPSC from somatic cells is essential for designing new approaches to improve reprogramming efficiency. Here we report the development and application of an integrated quantitative proteomics platform for investigating differences in protein expressions between mouse embryonic fibroblasts (MEF) and MEF-derived iPSC. This platform consists of 16O/18O labeling, multidimensional peptide separation coupled with tandem mass spectrometry, and data analysis with UNiquant software. Using this platform a total of 2,481 proteins were identified and quantified from the 16O/18O-labeled MEF-iPSC proteome mixtures with a false discovery rate of 0.01. Among them, 218 proteins were significantly upregulated, while 247 proteins were significantly downregulated in iPSC compared to MEF. Many nuclear proteins, including Hdac1, Dnmt1, Pcna, Ccnd1, Smarcc1, and subunits in DNA replication and RNA polymerase II complex were found to be enhanced in iPSC. Protein network analysis revealed that Pcna functions as a hub orchestrating complicated mechanisms including DNA replication, epigenetic inheritance (Dnmt1) and chromatin remodeling (Smarcc1) to reprogram MEF and maintain stemness of iPSC.

  20. Detection of gastrointestinal bleeding by means of Tc99m-labelled red cells - methodology and results

    Energy Technology Data Exchange (ETDEWEB)

    Bauer, R.; Kirchmann, K.; Flierdt, E. van de; Langhammer, H.R.; Pabst, H.W.

    1988-12-01

    Tc99m-labelled red cells were applied in 103 investigations of 91 patients to detect and localize occult gastrointestinal (GI) bleeding. In 60 examinations, GI-bleeding was proven. Correct localization of the bleeding site was achieved in 54 investigations. In 3 patients, bleeding from the lower intestinal tract could be positively excluded. These results were obtained in patients representing a negative selection, having undergone repeated endoscopy and angiography without positive result. 40% of all the bleeding sites were detected within 1 h. after tracer application, 25% only after a delay of 6 hrs. or more. To obtain good results, the whole investigation can become very time consuming and has to be performed according to a strict protocol. In addition, a tracer with high specificity to the intravasal space and high long-term stability has to be applied. Those requirements are met best by red cells labelled with Tc99m-according to a mixed in vivo/in vitro protocol.

  1. Fast, label-free super-resolution live-cell imaging using rotating coherent scattering (ROCS) microscopy.

    Science.gov (United States)

    Jünger, Felix; Olshausen, Philipp V; Rohrbach, Alexander

    2016-01-01

    Living cells are highly dynamic systems with cellular structures being often below the optical resolution limit. Super-resolution microscopes, usually based on fluorescence cell labelling, are usually too slow to resolve small, dynamic structures. We present a label-free microscopy technique, which can generate thousands of super-resolved, high contrast images at a frame rate of 100 Hertz and without any post-processing. The technique is based on oblique sample illumination with coherent light, an approach believed to be not applicable in life sciences because of too many interference artefacts. However, by circulating an incident laser beam by 360° during one image acquisition, relevant image information is amplified. By combining total internal reflection illumination with dark-field detection, structures as small as 150 nm become separable through local destructive interferences. The technique images local changes in refractive index through scattered laser light and is applied to living mouse macrophages and helical bacteria revealing unexpected dynamic processes. PMID:27465033

  2. Cell and brain tissue imaging of the flavonoid fisetin using label-free two-photon microscopy.

    Science.gov (United States)

    Krasieva, Tatiana B; Ehren, Jennifer; O'Sullivan, Thomas; Tromberg, Bruce J; Maher, Pamela

    2015-10-01

    Over the last few years, we have identified an orally active, novel neuroprotective and cognition-enhancing molecule, the flavonoid fisetin. Fisetin not only has direct antioxidant activity but it can also increase the intracellular levels of glutathione, the major intracellular antioxidant. Fisetin can also activate key neurotrophic factor signaling pathways. In addition, it has anti-inflammatory activity against microglia and astrocytes and inhibits the activity of lipoxygenases, thereby reducing the production of pro-inflammatory eicosanoids and their by-products. However, key questions about its targets and brain penetration remain. In this study, we used label-free two-photon microscopy of intrinsic fisetin fluorescence to examine the localization of fisetin in living nerve cells and the brains of living mice. In cells, fisetin but not structurally related flavonols with different numbers of hydroxyl groups, localized to the nucleoli suggesting that key targets of fisetin may reside in this organelle. In the mouse brain, following intraperitoneal injection and oral administration, fisetin rapidly distributed to the blood vessels of the brain followed by a slower dispersion into the brain parenchyma. Thus, these results provide further support for the effects of fisetin on brain function. In addition, they suggest that label-free two-photon microscopy may prove useful for studying the intracellular and tissue distribution of other intrinsically-fluorescent flavonoids.

  3. Fast, label-free super-resolution live-cell imaging using rotating coherent scattering (ROCS) microscopy

    Science.gov (United States)

    Jünger, Felix; Olshausen, Philipp V.; Rohrbach, Alexander

    2016-07-01

    Living cells are highly dynamic systems with cellular structures being often below the optical resolution limit. Super-resolution microscopes, usually based on fluorescence cell labelling, are usually too slow to resolve small, dynamic structures. We present a label-free microscopy technique, which can generate thousands of super-resolved, high contrast images at a frame rate of 100 Hertz and without any post-processing. The technique is based on oblique sample illumination with coherent light, an approach believed to be not applicable in life sciences because of too many interference artefacts. However, by circulating an incident laser beam by 360° during one image acquisition, relevant image information is amplified. By combining total internal reflection illumination with dark-field detection, structures as small as 150 nm become separable through local destructive interferences. The technique images local changes in refractive index through scattered laser light and is applied to living mouse macrophages and helical bacteria revealing unexpected dynamic processes.

  4. A Radiochemical Biotechnological Approach: Preliminary Study of Lactose Uptake Rate by Kefir Cells, Using 14C-labeled Lactose, in Anaerobic Fermentation

    Science.gov (United States)

    Golfinopoulos, A.; Soupioni, M.; Kanellaki, M.; Koutinas, A. A.

    2008-08-01

    The effect of initial lactose concentration on lactose uptake rate by kefir free cells, during the lactose fermentation, was studied in this work. For the investigation 14C-labelled lactose was used due to the fact that labeled and unlabeled molecules are fermented in the same way. The results illustrated lactose uptake rates are about up to two fold higher at lower initial ∘Bé densities as compared with higher initial ∘Bé densities.

  5. Resolution of heterogeneous fluorescence emission signals and decay lifetime measurement on fluorochrome-labeled cells by phase-sensitive FCM

    Energy Technology Data Exchange (ETDEWEB)

    Steinkamp, J.A.; Crissman, H.A.

    1993-01-01

    A phase-sensitive flow cytometer has been developed to resolve signals from heterogeneous fluorescence emission spectra and quantify fluorescence decay times on cells labeled with fluorescent dyes. This instrument combines flow cytometry (FCM) and fluorescence spectroscopy measurement principles to provide unique capabilities for making phase-resolved measurements on single cells in flow, while preserving conventional FCM measurement capabilities. Stained cells are analyzed as they pass through an intensity-modulated (sinusoid) laser excitation beam. Fluorescence is measured orthogonally using a s barrier filter to block scattered laser excitation light, and a photomultiplier tube detector output signals, which are shifted in phase from a reference signal and amplitude demodulated, are processed by phase-sensitive detection electronics to resolve signals from heterogeneous emissions and quantify decay lifetimes directly. The output signals are displayed as frequency distribution histograms and bivariate diagrams using a computer-based data acquisition system. Results have demonstrated signal phase shift, amplitude demodulation, and average measurement of fluorescence lifetimes on stained cells; a detection limit threshold of 300 to 500 fluorescein isothiocyanate (FITC); fluorescence measurement precision of 1.3% on alignment fluorospheres and 3.4% on propidium iodide (PI)-stained cells; the resolution of PI and FITC signals from cells stainedin combination with PI and FITC, based on differences in their decay lifetimes; and the ability to measure single decay nines by the two-phase, phase comparator, method.

  6. Resolution of heterogeneous fluorescence emission signals and decay lifetime measurement on fluorochrome-labeled cells by phase-sensitive FCM

    Energy Technology Data Exchange (ETDEWEB)

    Steinkamp, J.A.; Crissman, H.A.

    1993-02-01

    A phase-sensitive flow cytometer has been developed to resolve signals from heterogeneous fluorescence emission spectra and quantify fluorescence decay times on cells labeled with fluorescent dyes. This instrument combines flow cytometry (FCM) and fluorescence spectroscopy measurement principles to provide unique capabilities for making phase-resolved measurements on single cells in flow, while preserving conventional FCM measurement capabilities. Stained cells are analyzed as they pass through an intensity-modulated (sinusoid) laser excitation beam. Fluorescence is measured orthogonally using a s barrier filter to block scattered laser excitation light, and a photomultiplier tube detector output signals, which are shifted in phase from a reference signal and amplitude demodulated, are processed by phase-sensitive detection electronics to resolve signals from heterogeneous emissions and quantify decay lifetimes directly. The output signals are displayed as frequency distribution histograms and bivariate diagrams using a computer-based data acquisition system. Results have demonstrated signal phase shift, amplitude demodulation, and average measurement of fluorescence lifetimes on stained cells; a detection limit threshold of 300 to 500 fluorescein isothiocyanate (FITC); fluorescence measurement precision of 1.3% on alignment fluorospheres and 3.4% on propidium iodide (PI)-stained cells; the resolution of PI and FITC signals from cells stainedin combination with PI and FITC, based on differences in their decay lifetimes; and the ability to measure single decay nines by the two-phase, phase comparator, method.

  7. A pulse-chase strategy for EdU labelling assay is able to rapidly quantify cell division orientation.

    Science.gov (United States)

    Yin, Xiaofeng; Tsukaya, Hirokazu

    2016-09-01

    Measurement of the direction of cell division is an important, yet difficult, task to analyse how a plant organ acquires its final shape from an initially small group of cells. We introduce a method that rapidly and easily quantifies cell division direction and is applicable to all plant species. A pulse-chase strategy for 5-ethynyl-2'-deoxyuridine (EdU) labelling assay was established and was shown to be successful for leaves of Arabidopsis thaliana (Arabidopsis) and Juncus prismatocarpus. By optimization of the pulse and chase periods, most of the signals obtained were sets of daughter nuclei. For Arabidopsis, the optimal time was a 45-min pulse and a 7-h chase. For J. prismatocarpus, the optimal time was a 2-h pulse and a 13.5-h chase. The positions of the daughter nuclei were used to quantify cell division direction in the Arabidopsis leaf primordia. Overall, cell division along the proximal-distal axis was more frequent than along the medial-lateral axis. In petiole, major vein, minor vein and margin areas, the major cell division direction seemed to be coincident with the direction of auxin flow. The advantages of our method over the few methods used previously are discussed. We anticipate that it will provide opportunities to study plant development in the near future. PMID:27121010

  8. A pulse-chase strategy for EdU labelling assay is able to rapidly quantify cell division orientation.

    Science.gov (United States)

    Yin, Xiaofeng; Tsukaya, Hirokazu

    2016-09-01

    Measurement of the direction of cell division is an important, yet difficult, task to analyse how a plant organ acquires its final shape from an initially small group of cells. We introduce a method that rapidly and easily quantifies cell division direction and is applicable to all plant species. A pulse-chase strategy for 5-ethynyl-2'-deoxyuridine (EdU) labelling assay was established and was shown to be successful for leaves of Arabidopsis thaliana (Arabidopsis) and Juncus prismatocarpus. By optimization of the pulse and chase periods, most of the signals obtained were sets of daughter nuclei. For Arabidopsis, the optimal time was a 45-min pulse and a 7-h chase. For J. prismatocarpus, the optimal time was a 2-h pulse and a 13.5-h chase. The positions of the daughter nuclei were used to quantify cell division direction in the Arabidopsis leaf primordia. Overall, cell division along the proximal-distal axis was more frequent than along the medial-lateral axis. In petiole, major vein, minor vein and margin areas, the major cell division direction seemed to be coincident with the direction of auxin flow. The advantages of our method over the few methods used previously are discussed. We anticipate that it will provide opportunities to study plant development in the near future.

  9. Cytotoxic effects of 125I-labeled PBZr ligand PK 11195 in prostatic tumor cells: therapeutic implications

    International Nuclear Information System (INIS)

    The effect of [125I]PK 11195 was examined in human prostatic tumor cells (DU 145) in culture and compared with Na[125I] and non-radioactive PK 11195. [125I]PK 11195 was clearly cytocidal. The data for dose-related cell survival with [125I]PK 11195 showed a linear relationship. Na[125I] or non-labeled PK 11195 at similar concentrations did not lead to any cell killing. The uptake of [125I]PK 11195 and [3H]PK 11195 in cells was very similar. Fragmentation of DNA measured by agarose gel electrophoresis showed that exposure of DU 145 cells to [125I]PK 11195 for 1, 4 or 24 h caused no fragmentation. These results indicate that nuclear DNA is not the prime binding site for [125I]PK 11195, which is consistent with the presence of specific peripheral benzodiazepine receptors (PBZr) in the mitochondria. The cell killing effect of [125I]PK 11195 suggests the use of PBZr ligand for radiotherapy

  10. Study of acetowhitening mechanisms in live mammalian cells with label-free subcellular-level multimodal nonlinear optical microscopy

    Science.gov (United States)

    Lin, Jian; Teh, Sengkhoon; Zheng, Wei; Wang, Zi; Huang, Zhiwei

    2015-03-01

    The tissue acetowhitening effect in acetic acid instillation procedure is a simple and economic method for neoplasia detection and has been clinically utilized since 1925. It is suspected that the optical property (e.g. scattering) change in acetowhitening is due to coagulation of intracellular proteins, but no experimental proof has been reported yet. In this work, we use third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) to investigate the acetowhitening phenomenon induced by acidic acid in live mammalian cells without labeling. We studied the acetowhitening effect with different acetic acid concentrations and the co-localized TPEF and THG imaging on tryptophan and NADH at subcellular-level reveals that the acetowhitening phenomenon is highly related with proteins involved in metabolic pathways in the nucleus and cytoplasm in live cells.

  11. Pharmacokinetics, tissue distribution, and cell localization of [35S]methionine-labeled recombinant human and murine alpha interferons in mice

    International Nuclear Information System (INIS)

    The pharmacokinetics, tissue distribution, cell localization, and penetration into tumor xenografts of recombinant [35S]methionine-labeled human alpha interferon (HuIFN-alpha) and murine alpha interferon (MuIFN-alpha) were examined in mice. Both interferons (IFNs) were removed from the blood in a rapid biphasic manner; HuIFN-alpha was cleared faster than MuIFN-alpha. Tissues were analyzed for radioactivity and over 90% of the IFNs was accounted for. The IFNs were detected predominantly in liver, kidney, gastrointestinal tract, pancreas, spleen, and lung. The levels of MuIFN-alpha compared with HuIFN-alpha were greater in the liver, spleen, and lung and less in the kidney, pancreas, and gastrointestinal tract. Heart, brain, testes, thymus, lymph nodes, fat, skin, and skeletal muscle contained much lower but measurable levels of both IFNs. There was penetration of HuIFN-alpha into tumor xenografts. The pharmacokinetics of IFN-alpha were independent of the strain of mouse, BALB/c or CBA, immune deprivation, or the presence of a tumor xenograft. Autoradiography of tissue sections from mice given injections of HuIFN-alpha or MuIFN-alpha indicated focal radioactivity in proximal convoluted tubules in the kidney and diffuse radioactivity in the liver, gastrointestinal tract, and pancrease. MuIFN-alpha, but not HuIFN-alpha, showed intense localization in cells in hepatic sinusoids, marginal zones in the spleen, and pulmonary alveolar walls, suggesting uptake by cells of the monocyte/macrophage lineage in these sites. The study shows the utility of biosynthetic labeling for pharmacokinetic studies of cytokines, clear differences in tissue distribution of IFN-alpha according to its species of origin, and targeting of homologous IFN-alpha to cells of the monocytic lineage

  12. Label-free assessment of adipose-derived stem cell differentiation using coherent anti-Stokes Raman scattering and multiphoton microscopy

    OpenAIRE

    Mouras, Rabah; Bagnaninchi, Pierre O.; Downes, Andrew R; Elfick, Alistair P D

    2012-01-01

    ABSTRACT. Adult stem cells (SCs) hold great potential as likely candidates for disease therapy but also as sources of differentiated human cells in vitro models of disease. In both cases, the label-free assessment of SC differentiation state is highly desirable, either as a quality-control technology ensuring cells to be used clinically are of the desired lineage or to facilitate in vitro time-course studies of cell differentiation. We investigate the potential of nonlinear optical microscopy...

  13. Experimental bladder regeneration using a poly-L-lactide/silk fibroin scaffold seeded with nanoparticle-labeled allogenic bone marrow stromal cells

    Directory of Open Access Journals (Sweden)

    Yudintceva NM

    2016-09-01

    Full Text Available Natalia M Yudintceva,1 Yulia A Nashchekina,1,2 Miralda I Blinova,1 Nadezhda V Orlova,3 Alexandr N Muraviov,3 Tatiana I Vinogradova,3 Magomed G Sheykhov,3 Elena Y Shapkova,3 Dmitriy V Emeljannikov,3 Petr K Yablonskii,3,4 Igor A Samusenko,5 Anastasiya L Mikhrina,6 Artem V Pakhomov,7 Maxim A Shevtsov1,7,8 1Department of Cell Culture, Institute of Cytology of the Russian Academy of Sciences (RAS, 2Nanotechnology and Telecommunications, Institute of Physics, Peter the Great St Petersburg Polytechnic University, 3Department of Urology, Federal State Institution Saint Petersburg Research Institute of Phthisiopulmonology, Ministry of Health of Russia, 4Faculty of Medicine, Federal State Budgetary Institute, 5Department of Pathology, Federal State Budgetary Institute “Nikiforov Russian Centre of Emergency and Radiation Medicine” of the Ministry of Health of Russia, 6Department of Pathomorphology, I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Science, 7Department of Radiology, Federal Almazov North-West Medical Research Center, 8Department of Experimental Medicine, First I.P. Pavlov State Medical University of St Petersburg, St Petersburg, Russia Abstract: In the present study, a poly-L-lactide/silk fibroin (PL-SF bilayer scaffold seeded with allogenic bone marrow stromal cells (BMSCs was investigated as a potential approach for bladder tissue engineering in a model of partial bladder wall cystectomy in rabbits. The inner porous layer of the scaffold produced from silk fibroin was designed to promote cell proliferation and the outer layer produced from poly-L-lactic acid to serve as a waterproof barrier. To compare the feasibility and efficacy of BMSC application in the reconstruction of bladder defects, 12 adult male rabbits were divided into experimental and control groups (six animals each that received a scaffold seeded with BMSCs or an acellular one, respectively. For BMSC tracking in the graft in

  14. The Effect of Superparamagnetic Iron Oxide with iRGD Peptide on the Labeling of Pancreatic Cancer Cells In Vitro: A Preliminary Study

    Directory of Open Access Journals (Sweden)

    Hou Dong Zuo

    2014-01-01

    Full Text Available The iRGD peptide loaded with iron oxide nanoparticles for tumor targeting and tissue penetration was developed for targeted tumor therapy and ultrasensitive MR imaging. Binding of iRGD, a tumor homing peptide, is mediated by integrins, which are widely expressed on the surface of cells. Several types of small molecular drugs and nanoparticles can be transfected into cells with the help of iRGD peptide. Thus, we postulate that SPIO nanoparticles, which have good biocompatibility, can also be transfected into cells using iRGD. Despite the many kinds of cell labeling studies that have been performed with SPIO nanoparticles and RGD peptide or its analogues, only a few have applied SPIO nanoparticles with iRGD peptide in pancreatic cancer cells. This paper reports our preliminary findings regarding the effect of iRGD peptide (CRGDK/RGPD/EC combined with SPIO on the labeling of pancreatic cancer cells. The results suggest that SPIO with iRGD peptide can enhance the positive labeling rate of cells and the uptake of SPIO. Optimal functionalization was achieved with the appropriate concentration or concentration range of SPIO and iRGD peptide. This study describes a simple and economical protocol to label panc-1 cells using SPIO in combination with iRGD peptide and may provide a useful method to improve the sensitivity of pancreatic cancer imaging.

  15. Translational research on reprogramming of somatic cells

    Institute of Scientific and Technical Information of China (English)

    Yanhua Li; Jiahui Yin; Bingbing Zhang; Ping Zhou; Bin Feng; Fangyi Zhang; Yongzhong Lin; Zhanhua Liang; Jianling Du; Minghui Lü; Tiezheng Zheng; Jie Lin; Siyu Liu; Hao Hong; Xing Meng; Dandan Xia; Yang Sun; Pan Wei; Nan Cai; Hongye Li; Shuang Wu; Hui Zhao; Changkai Sun; Yuyuan Li; Changyu Gao; Wei Li; Ye Dai; Junde Wang; Hui Zhao; Xiaoxin Tan; Lili Men; Hui Ma; Jun Xu; Xiaohan Yang; Zengchun Hu; Ling Wang; Hong Wang; Pin Sun; Huifang Guo; Guirong Song; Hui Liu1; Baoshuai Shan; Lu Han; Linlang Liang; Min Wang; Xiaochen Wang; Dan Wang; Guihua Chen; Jianting Chen; Xiangyou Sun; Jun Xue; Zhiqi Wang; Jing Wang; Yongqing Zhang; Dongfeng Cai; Mozhen Liu; Guiping Zhang; Guoming Luan; Jianli Wang; Ming Fan; Xuetao Cao; Chao Wan; Qigui Liu; Anchun Yin

    2014-01-01

    Cerebrovascular diseases,dementia,diabetes,malignant tumors and degenerative bone diseases remain high prevalence,incidence,disability and mortality rates.One important reason might be the slow or stagnated progress in translating and applying cytoprotection and cellular repair researches into clinical practice.Based on collaboration among biomedical re-searchers,database experts,computer programmers,statisticians and management engineers,this is the first study to apply quanti-tative comparison on the overall characteristics and partial correlation analysis on the large-scale complex information and data regarding the topic“mature cells can be reprogrammed to become pluripotent”proposed by Sir John B.Gurdon and Shinya Yamanaka who were jointly awarded with 2012 Nobel Prize in Physiology or Medicine,as well as articles that cited publications of the two Nobel Laureates to discuss the prospects of translating somatic cell reprogramming researches into clinical practice and cor-responding implementation strategies.The study found that there was statistically significant difference between the two Nobel Laureates with regard to the number,publication date,subject categories and scientific and technological focuses of their origi-nal researches.The study revealed the importance,objectives,approaches and research trends of translational medicine,especially translational neuroscience.The study also identified the challenges that China should overcome to improve its medical research management scheme.

  16. Fully automated TV-image analysis of the cell-cycle: comparison of the PLM method with determinations of the percentage and the DNA content of labelled cells.

    Science.gov (United States)

    Wachsmuth, E D; van Golitschek, M; Macht, F; Maurer-Schultze, B

    1988-01-01

    A cell-cycle analysis based on a fully automated TV-image scanning system is proposed to replace the laborious PLM method. To compare the efficiency of the two procedures, cell-cycle parameters were assessed in Ehrlich (diploid and hyperdiploid), L-1210, and JB-1 mouse ascites tumours and in rat jejunal crypts. The percentages of labelled mitoses (PLM) were counted visually on Feulgen-stained autoradiographs obtained at various times after a single 3H-thymidine pulse. The fraction of labelled cells (P) and the DNA ratio of labelled and unlabelled cells were measured by TV-image analysis in the same slides and plotted against time. Within practical limits, TV-image analysis using the P-curve gives the same results as the PLM method. Using the P-curve has the important advantage that its first part, beginning at the time of 3H-thymidine injection and ending at the first maximum, furnishes more information about the cell cycle than the corresponding part of the PLM curve. It can be used to compute tG2M tS and the ratio of the growth faction index to the cell-cycle time (IP/tC) whereas the first part of the PLM-curve reveals only the length of the S-phase (tS). The IP/tC ratio is a readily accessible measure of growth and increases when the cells divide more frequently. Cell death rates may be neglected since the ratio is determined within less than the duration of one cell cycle. Moreover, the data from the first part of the P curve indicate whether there is a large non-growth fraction. If the non-growth fraction is small, i.e. if IP approximately 1, the P curve need only be measured until the first maximum is reached so that fewer samples and animals are required. If the non-growth fraction is large or unknown, the cell-cycle parameters are calculated by reference to the position and size not only of the first minimum and the first maximum, but also of the second minimum of the P curve.

  17. The production of intrinsically labeled milk and meat protein is feasible and provides functional tools for human nutrition research

    NARCIS (Netherlands)

    Pennings, B.; Pellikaan, W.F.; Senden, J.M.G.; Vuuren, van A.M.; Sikkema, J.; Loon, van L.J.C.

    2011-01-01

    Administration of labeled, free amino acids does not allow direct assessment of in vivo dietary protein digestion and absorption kinetics. Consequently, dietary protein sources with labeled amino acids incorporated within their protein matrix are required. The aim of the present study was to produce

  18. The radioactive labeling of monocytes

    International Nuclear Information System (INIS)

    With the aim of studying a possible relationship between circulating monocytes and Sternberg-Reed cells investigations were started on the specific labeling of monocytes. In this thesis the literature on the pertinent data has been reviewed and a series of experiments on the monocyte labeling procedure has been described. The principles of cell labeling with radioactive compounds were discussed. 1. Total separation of the particular cell population to be labeled and subsequent labeling with a non-specific radiopharmaceutical. 2. Specific cell labeling in a mixture of cell types based on a well defined affinity of the cell under study for the radiopharmaceutical used. Next the radionuclides that can be used for cell labeling purposes were discussed with special attention for 111In and its chelates. The principles of radiodosimetry were also discussed shortly. This section was focussed on the radiation dose the labeled cells receive because of the intracellular localized radioactivity. The radiation burden is high in comparison to amounts of radiation known to affect cell viability. A newly developed method for labeling monocytes specifically by phagocytosis of 111In-Fe-colloid without apparent loss of cells was described in detail. (Auth.)

  19. Usefulness of PKH fluorescent labelling to study leukemic cell proliferation with various cytostatic drugs or acetyl tetrapeptide – AcSDKP

    International Nuclear Information System (INIS)

    PKH67 labelling was compared for classical proliferation assessment (using S phase evaluation) to analyse the cell proliferation of 29 AML patients treated or not with various drugs. Among these drugs, the effect of tetrapeptide AcSDKP or AcSDKP-NH2 on AML cells, stimulated or not by cytokines, was also evaluated in order to determine (i) if AcSDKP was able to inhibit blast cell proliferation as it inhibits haematopoietic progenitors (ii) if AcSDKP-NH2 was more stable than AcSDKP with FBS. For PKH labeling, cells were suspended in Diluent C, and rapidly admixed with PKH67 solution at 20 μM PKH67. Staining was stopped by addition of FBS. A good correlation between PKH67 labelling and bromodeoxyuridine incorporation was obtained first with 6/9 patients for control cells, then for 11/17 AML patients treated with classical antileukemic drugs (among whom 4 were also treated with AcSDKP). The effect of AcSDKP was also studied on 7 patients. The discrepancy between both methods was essentially due to an accumulation of cells into different cycle phases measured by BrdUrd incorporation secondary to drug action and PKH67 labelling which measured the dynamic proliferation. This last method allows identifying resistant cells which still proliferate. AcSDKP or AcSDKP-NH2 induced a decrease of leukemic cell proliferation in 5/7 patients when cytokines were added (in order to stimulate proliferation) one day after tetrapeptide AcSDKP or AcSDKP-NH2. No effect on proliferation was noted when cytokines were added to AcSDKP-NH2. PKH67 labelling method is a powerful tool for cell proliferation assessment in patients with AML, even in cells treated by various drugs

  20. Systematically labeling developmental stage-specific genes for the study of pancreatic β-cell differentiation from human embryonic stem cells.

    Science.gov (United States)

    Liu, Haisong; Yang, Huan; Zhu, Dicong; Sui, Xin; Li, Juan; Liang, Zhen; Xu, Lei; Chen, Zeyu; Yao, Anzhi; Zhang, Long; Zhang, Xi; Yi, Xing; Liu, Meng; Xu, Shiqing; Zhang, Wenjian; Lin, Hua; Xie, Lan; Lou, Jinning; Zhang, Yong; Xi, Jianzhong; Deng, Hongkui

    2014-10-01

    The applications of human pluripotent stem cell (hPSC)-derived cells in regenerative medicine has encountered a long-standing challenge: how can we efficiently obtain mature cell types from hPSCs? Attempts to address this problem are hindered by the complexity of controlling cell fate commitment and the lack of sufficient developmental knowledge for guiding hPSC differentiation. Here, we developed a systematic strategy to study hPSC differentiation by labeling sequential developmental genes to encompass the major developmental stages, using the directed differentiation of pancreatic β cells from hPSCs as a model. We therefore generated a large panel of pancreas-specific mono- and dual-reporter cell lines. With this unique platform, we visualized the kinetics of the entire differentiation process in real time for the first time by monitoring the expression dynamics of the reporter genes, identified desired cell populations at each differentiation stage and demonstrated the ability to isolate these cell populations for further characterization. We further revealed the expression profiles of isolated NGN3-eGFP(+) cells by RNA sequencing and identified sushi domain-containing 2 (SUSD2) as a novel surface protein that enriches for pancreatic endocrine progenitors and early endocrine cells both in human embryonic stem cells (hESC)-derived pancreatic cells and in the developing human pancreas. Moreover, we captured a series of cell fate transition events in real time, identified multiple cell subpopulations and unveiled their distinct gene expression profiles, among heterogeneous progenitors for the first time using our dual reporter hESC lines. The exploration of this platform and our new findings will pave the way to obtain mature β cells in vitro. PMID:25190258

  1. Development and experimental basis of local subretinal technique of xenogenic’s injection stem cells labelled by magnetic perticles

    Directory of Open Access Journals (Sweden)

    Yu. A. Belyy

    2014-10-01

    Full Text Available Purpose: is to develop a technique for local subretinal injection of xenogeneic stem cells labeled with magnetic particles and to prove experimentally its effectiveness.Material and methods: We used a line of stem cells HEK-293 GFP,labeled with magnetic particles. The study was made on 84 eyes of 42 chinchilla rabbits 6 months of age, the weight were from 2.5 to 3.5 kg. All right eyes were experimental (42 eyes and all left eyes (42 eyes were the control group. In the experimental group we used original complex of polymer elastic magnetic implant (PEMI with laser probe and fixed it to the sclera, then we made a median vitrectomy and injected HEK-293 GFP under the retina using a specially designed dispenser. In the control group PEMI was not fixed. We examined animals using biomicroscopy, ophthalmoscopy, ultrasound scanning, optical coherence tomography  OCT, computer tomography (CT, morphological study (cryohistological sections in 1, 3, 5, 7, 14 day and 1 month after surgery.Results: According the results of biomicroscopy in observation periods up to 3 days the vascular injection was visualized in the area operation. According the results of ophthalmoscopy and ultrasound scanning in 1 day the local retinal detachment was visualized in the area of local injection of the stem cells, which was not visualized in terms of further observations. CT helped us to confirm the local place of PEMI fixation. The morphological study results showed that cells were located in the subretinal space up to 14 days in the experimental group, and only up 3 days in the control group.Conclusion: The suggested surgical technique enables to control the injection of cells into the subretinal space, reduces the risk of tissue damage and exit cells in the vitreous space. The suggested methodology allows the fixing of the cellular material in the local place of the injection and enables to predict cells`s movement.

  2. Live-Cell, Label-Free Identification of GABAergic and Non-GABAergic Neurons in Primary Cortical Cultures Using Micropatterned Surface

    Science.gov (United States)

    Kono, Sho; Kushida, Takatoshi; Hirano-Iwata, Ayumi; Niwano, Michio; Tanii, Takashi

    2016-01-01

    Excitatory and inhibitory neurons have distinct roles in cortical dynamics. Here we present a novel method for identifying inhibitory GABAergic neurons from non-GABAergic neurons, which are mostly excitatory glutamatergic neurons, in primary cortical cultures. This was achieved using an asymmetrically designed micropattern that directs an axonal process to the longest pathway. In the current work, we first modified the micropattern geometry to improve cell viability and then studied the axon length from 2 to 7 days in vitro (DIV). The cell types of neurons were evaluated retrospectively based on immunoreactivity against GAD67, a marker for inhibitory GABAergic neurons. We found that axons of non-GABAergic neurons grow significantly longer than those of GABAergic neurons in the early stages of development. The optimal threshold for identifying GABAergic and non-GABAergic neurons was evaluated to be 110 μm at 6 DIV. The method does not require any fluorescence labelling and can be carried out on live cells. The accuracy of identification was 98.2%. We confirmed that the high accuracy was due to the use of a micropattern, which standardized the development of cultured neurons. The method promises to be beneficial both for engineering neuronal networks in vitro and for basic cellular neuroscience research. PMID:27513933

  3. Clinical utility of labeled cells for detection of allograft rejection and myocardial infarction

    Energy Technology Data Exchange (ETDEWEB)

    Fawwaz, R.A.

    1984-07-01

    The choice of a specific radiolabeled blood component for use in detection of allograft rejection depends on several factors including the immunosuppressive agents used, the type of organ allografted, and particularly the length of time the allograft resides in the host and the duration of rejection. To date, only the use of 111In-labeled platelets in renal allograft recipients immunosuppressed with azathioprine and corticosteroids has shown clinical promise in the detection of early allograft rejection. Radiolabeled blood components are unlikely to play a significant role in detection of myocardial infarction. The use of these agents for monitoring therapeutic interventions or as indicators of prognosis in patients with myocardial infarction continues to be investigated.

  4. Proximity-Directed Labeling Reveals a New Rapamycin-Induced Heterodimer of FKBP25 and FRB in Live Cells

    Science.gov (United States)

    2016-01-01

    Mammalian target of rapamycin (mTOR) signaling is a core pathway in cellular metabolism, and control of the mTOR pathway by rapamycin shows potential for the treatment of metabolic diseases. In this study, we employed a new proximity biotin-labeling method using promiscuous biotin ligase (pBirA) to identify unknown elements in the rapamycin-induced interactome on the FK506-rapamycin binding (FRB) domain in living cells. FKBP25 showed the strongest biotin labeling by FRB–pBirA in the presence of rapamycin. Immunoprecipitation and immunofluorescence experiments confirmed that endogenous FKBP25 has a rapamycin-induced physical interaction with the FRB domain. Furthermore, the crystal structure of the ternary complex of FRB–rapamycin–FKBP25 was determined at 1.67-Å resolution. In this crystal structure we found that the conformational changes of FRB generate a hole where there is a methionine-rich space, and covalent metalloid coordination was observed at C2085 of FRB located at the bottom of the hole. Our results imply that FKBP25 might have a unique physiological role related to metallomics in mTOR signaling. PMID:27610411

  5. Proximity-Directed Labeling Reveals a New Rapamycin-Induced Heterodimer of FKBP25 and FRB in Live Cells.

    Science.gov (United States)

    Lee, Song-Yi; Lee, Hakbong; Lee, Hye-Kyeong; Lee, Seung-Won; Ha, Sung Chul; Kwon, Taejoon; Seo, Jeong Kon; Lee, Changwook; Rhee, Hyun-Woo

    2016-08-24

    Mammalian target of rapamycin (mTOR) signaling is a core pathway in cellular metabolism, and control of the mTOR pathway by rapamycin shows potential for the treatment of metabolic diseases. In this study, we employed a new proximity biotin-labeling method using promiscuous biotin ligase (pBirA) to identify unknown elements in the rapamycin-induced interactome on the FK506-rapamycin binding (FRB) domain in living cells. FKBP25 showed the strongest biotin labeling by FRB-pBirA in the presence of rapamycin. Immunoprecipitation and immunofluorescence experiments confirmed that endogenous FKBP25 has a rapamycin-induced physical interaction with the FRB domain. Furthermore, the crystal structure of the ternary complex of FRB-rapamycin-FKBP25 was determined at 1.67-Å resolution. In this crystal structure we found that the conformational changes of FRB generate a hole where there is a methionine-rich space, and covalent metalloid coordination was observed at C2085 of FRB located at the bottom of the hole. Our results imply that FKBP25 might have a unique physiological role related to metallomics in mTOR signaling. PMID:27610411

  6. Magnetoplex based on MnFe{sub 2}O{sub 4} nanocrystals for magnetic labeling and MR imaging of human mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Jaemoon [Yonsei University, Department of Radiology, College of Medicine (Korea, Republic of); Lim, Eun-Kyung [Yonsei University, Department of Chemical and Biomolecular Engineering (Korea, Republic of); Lee, Eun-Sook; Suh, Jin-Suck [Yonsei University, Department of Radiology, College of Medicine (Korea, Republic of); Haam, Seungjoo, E-mail: haam@yonsei.ac.k [Yonsei University, Department of Chemical and Biomolecular Engineering (Korea, Republic of); Huh, Yong-Min, E-mail: ymhuh@yumc.yonsei.ac.k [Yonsei University, Department of Radiology, College of Medicine (Korea, Republic of)

    2010-05-15

    For efficient labeling and tracking via magnetic resonance (MR) imaging of human mesenchymal stem cells (h-MSCs), magnetic labeling agents must be responsive to an external magnetic field. Thus, we developed ultrasensitive magnetoplex as a magnetic labeling agent composed of PEGylated MnFe{sub 2}O{sub 4} nanocrystals (PMNCs) and polycationics (poly-l-lysine, PLL) for efficient labeling of the h-MSCs and monitoring of the transplanted h-MSCs for a long term. PMNCs were prepared by nanoemulsion methods composed of MnFe{sub 2}O{sub 4} nanocrystals (MNCs) and amphiphilic polymers (mPEG-dodecanoic acid). The prepared PMNCs exhibited excellent biocompatibility and their polycationic complexes (PMNCs/PLL) demonstrated remarkable sensitivity compared with magnetic iron oxide nanoparticles (MION)/PLL or Ferumoxides/PLL. Furthermore, PMNCs demonstrated the potentials for novel diagnostic and therapeutic strategies with potential applications in various biomedical fields.

  7. Paramagnetic particles carried by cell-penetrating peptide tracking of bone marrow mesenchymal stem cells, a research in vitro

    International Nuclear Information System (INIS)

    The ability to track the distribution and differentiation of stem cells by high-resolution imaging techniques would have significant clinical and research implications. In this study, a model cell-penetrating peptide was used to carry gadolinium particles for magnetic resonance imaging of the mesenchymal stem cells. The mesenchymal stem cells were isolated from rat bone marrow by Percoll and identified by osteogenic differentiation in vitro. The cell-penetrating peptides labeled with fluorescein-5-isothiocyanate and gadolinium were synthesized by a solid-phase peptide synthesis method and the relaxivity of cell-penetrating peptide-gadolinium paramagnetic conjugate on 400 MHz nuclear magnetic resonance was 5.7311 ± 0.0122 mmol-1 s-1, higher than that of diethylenetriamine pentaacetic acid gadolinium (p < 0.05). Fluorescein imaging confirmed that this new peptide could internalize into the cytoplasm and nucleus. Gadolinium was efficiently internalized into mesenchymal stem cells by the peptide in a time- or concentration-dependent fashion, resulting in intercellular T1 relaxation enhancement, which was obviously detected by 1.5 T magnetic resonance imaging. Cytotoxicity assay and flow cytometric analysis showed the intercellular contrast medium incorporation did not affect cell viability and membrane potential gradient. The research in vitro suggests that the newly constructed peptides could be a vector for tracking mesenchymal stem cells

  8. A semi-automated technique for labeling and counting of apoptosing retinal cells

    OpenAIRE

    Bizrah, M.; Dakin, S C; Guo, L.; F. Rahman1; Parnell, M.; Normando, E.; Nizari, S; Davis, B; Younis, A.; Cordeiro, M F

    2014-01-01

    Background Retinal ganglion cell (RGC) loss is one of the earliest and most important cellular changes in glaucoma. The DARC (Detection of Apoptosing Retinal Cells) technology enables in vivo real-time non-invasive imaging of single apoptosing retinal cells in animal models of glaucoma and Alzheimer’s disease. To date, apoptosing RGCs imaged using DARC have been counted manually. This is time-consuming, labour-intensive, vulnerable to bias, and has considerable inter- and intra-operator varia...

  9. Fabrication of gold nanodot arrays on a transparent substrate as a nanobioplatform for label-free visualization of living cells

    International Nuclear Information System (INIS)

    Two-dimensional gold (Au) nanodot arrays on a transparent substrate were fabricated for imaging of living cells. A nanoporous alumina mask with large-area coverage capability was prepared by a two-step chemical wet etching process after a second anodization. Highly ordered Au nanodot arrays were formed on indium-tin-oxide (ITO) glass using very thin nanoporous alumina of approximately 200 nm thickness as an evaporation mask. The large-area Au nanodot arrays on ITO glass were modified with RGD peptide (arginine; glycine; aspartic acid) containing a cysteine (Cys) residue and then used to immobilize human cancer HeLa cells, the morphology of which was observed by confocal microscopy. The confocal micrographs of living HeLa cells on Au nanodot arrays revealed enhanced contrast and resolution, which enabled discernment of cytoplasmic organelles more clearly. These results suggest that two-dimensional Au nanodot arrays modified with RGD peptide on ITO glass have potential as a biocompatible nanobioplatform for the label-free visualization and adhesion of living cells.

  10. Label-free capture of breast cancer cells spiked in buffy coats using carbon nanotube antibody micro-arrays

    Science.gov (United States)

    Khosravi, Farhad; Trainor, Patrick; Rai, Shesh N.; Kloecker, Goetz; Wickstrom, Eric; Panchapakesan, Balaji

    2016-04-01

    We demonstrate the rapid and label-free capture of breast cancer cells spiked in buffy coats using nanotube-antibody micro-arrays. Single wall carbon nanotube arrays were manufactured using photo-lithography, metal deposition, and etching techniques. Anti-epithelial cell adhesion molecule (EpCAM) antibodies were functionalized to the surface of the nanotube devices using 1-pyrene-butanoic acid succinimidyl ester functionalization method. Following functionalization, plain buffy coat and MCF7 cell spiked buffy coats were adsorbed on to the nanotube device and electrical signatures were recorded for differences in interaction between samples. A statistical classifier for the ‘liquid biopsy’ was developed to create a predictive model based on dynamic time warping to classify device electrical signals that corresponded to plain (control) or spiked buffy coats (case). In training test, the device electrical signals originating from buffy versus spiked buffy samples were classified with ˜100% sensitivity, ˜91% specificity and ˜96% accuracy. In the blinded test, the signals were classified with ˜91% sensitivity, ˜82% specificity and ˜86% accuracy. A heatmap was generated to visually capture the relationship between electrical signatures and the sample condition. Confocal microscopic analysis of devices that were classified as spiked buffy coats based on their electrical signatures confirmed the presence of cancer cells, their attachment to the device and overexpression of EpCAM receptors. The cell numbers were counted to be ˜1-17 cells per 5 μl per device suggesting single cell sensitivity in spiked buffy coats that is scalable to higher volumes using the micro-arrays.

  11. Label-free capture of breast cancer cells spiked in buffy coats using carbon nanotube antibody micro-arrays.

    Science.gov (United States)

    Khosravi, Farhad; Trainor, Patrick; Rai, Shesh N; Kloecker, Goetz; Wickstrom, Eric; Panchapakesan, Balaji

    2016-04-01

    We demonstrate the rapid and label-free capture of breast cancer cells spiked in buffy coats using nanotube-antibody micro-arrays. Single wall carbon nanotube arrays were manufactured using photo-lithography, metal deposition, and etching techniques. Anti-epithelial cell adhesion molecule (EpCAM) antibodies were functionalized to the surface of the nanotube devices using 1-pyrene-butanoic acid succinimidyl ester functionalization method. Following functionalization, plain buffy coat and MCF7 cell spiked buffy coats were adsorbed on to the nanotube device and electrical signatures were recorded for differences in interaction between samples. A statistical classifier for the 'liquid biopsy' was developed to create a predictive model based on dynamic time warping to classify device electrical signals that corresponded to plain (control) or spiked buffy coats (case). In training test, the device electrical signals originating from buffy versus spiked buffy samples were classified with ∼100% sensitivity, ∼91% specificity and ∼96% accuracy. In the blinded test, the signals were classified with ∼91% sensitivity, ∼82% specificity and ∼86% accuracy. A heatmap was generated to visually capture the relationship between electrical signatures and the sample condition. Confocal microscopic analysis of devices that were classified as spiked buffy coats based on their electrical signatures confirmed the presence of cancer cells, their attachment to the device and overexpression of EpCAM receptors. The cell numbers were counted to be ∼1-17 cells per 5 μl per device suggesting single cell sensitivity in spiked buffy coats that is scalable to higher volumes using the micro-arrays.

  12. Therapeutics with SPION-labeled stem cells for the main diseases related to brain aging: a systematic review

    Directory of Open Access Journals (Sweden)

    Alvarim LT

    2014-08-01

    Full Text Available Larissa T Alvarim,1,3,* Leopoldo P Nucci,2,* Javier B Mamani,1 Luciana C Marti,1 Marina F Aguiar,1,2 Helio R Silva,1,3 Gisele S Silva,1 Mariana P Nucci-da-Silva,4 Elaine A DelBel,5,6 Lionel F Gamarra1–31Hospital Israelita Albert Einstein, São Paulo, Brazil; 2Universidade Federal de São Paulo, UNIFESP, São Paulo, Brazil; 3Faculdade de Ciências Médicas da Santa Casa de São Paulo, São Paulo, Brazil; 4Departamento de Radiologia, Hospital das Clínicas, Universidade de São Paulo, Brazil; 5Universidade de São Paulo-Faculdade de Odontologia de Ribeirão Preto, São Paulo, Brazil; 6NAPNA- Núcleo de Apoio a Pesquisa em Neurociências Aplicadas, São Paulo, Brazil*These authors contributed equally to this workAbstract: The increase in clinical trials assessing the efficacy of cell therapy for structural and functional regeneration of the nervous system in diseases related to the aging brain is well known. However, the results are inconclusive as to the best cell type to be used or the best methodology for the homing of these stem cells. This systematic review analyzed published data on SPION (superparamagnetic iron oxide nanoparticle-labeled stem cells as a therapy for brain diseases, such as ischemic stroke, Parkinson’s disease, amyotrophic lateral sclerosis, and dementia. This review highlights the therapeutic role of stem cells in reversing the aging process and the pathophysiology of brain aging, as well as emphasizing nanotechnology as an important tool to monitor stem cell migration in affected regions of the brain.Keywords: iron oxide, dementia, stem cell, stroke, Parkinson’s disease, sclerosis disease, brain aging

  13. Labelling, quality control and clinical evaluation of monoclonal antibodies for scintigraphy. Final report of a co-ordinated research programme 1991-1996

    International Nuclear Information System (INIS)

    Realizing the potential of labelled monoclonal antibodies for in vivo diagnosis and therapy and the interest in many developing Member States for acquiring expertise in this field the IAEA initiated a co-ordinated research programme in 1991 focusing on 99Tcm labelling of antibodies, their quality control and scintigraphic evaluation. Twelve laboratories from Asia, Latin America, Europe and North America participated in this programme which was concluded in 1996. During this programme the participants investigated the 99Tcm labelling of a murine anti-CEA antibody using the method of chelating 99Tcm with the free sulfhydryl groups generated by reaction with reducing agents such as mercapto ethanol. During the later part of the programme this method was also extended to 99Tcm labelling of hIgG. All the participating laboratories could gain valuable experience in 99Tcm antibody labelling techniques and formulation of kits. Many of them have been use in patients by collaborating nuclear medicine specialists with satisfactory results. This report is a compilation of the detailed results obtained by the participating laboratories and includes a summary and assessment of the achievement of the CRP

  14. Effect of an Arctium lappa (burdock) extract on the labeling of blood constituents with technetium-99m and on the morphology of the red blood cells

    OpenAIRE

    Rosane de Figueiredo Neves; Silvana Ramos Farias Moreno; Bernardo Machado Rebello; Luiz Querino de Araújo Caldas; Adenilson de Souza da Fonseca; Mario Bernardo-Filho; Aldo da Cunha Medeiros

    2007-01-01

    Arctium lappa (burdock) has been used to treat inflammatory processes. Blood constituents labeled with technetium-99m (99mTc) have been utilized in nuclear medicine. It was evaluated the influence of a burdock extract on the labeling of blood constituents with 99mTc and on the morphometry of red blood cells. Blood samples from Wistar rats were incubated with burdock extract and the radiolabeling procedure was carried out. Plasma and blood cells, soluble and insoluble fractions of plasma and b...

  15. Labelling of T cell subsets under field conditions in tropical countries. Adaptation of the immuno-alkaline phosphatase staining method for blood smears

    DEFF Research Database (Denmark)

    Lisse, I M; Whittle, H; Aaby, P;

    1990-01-01

    Immuno-alkaline phosphatase (AP) staining for T cell subsets (CD4 and CD8) of smears from fingerprick blood functioned well under tropical climatic conditions when smears were stored frozen with silica gel before being labelled. Unlabelled smears were stored for up to 12 months and could be trans......Immuno-alkaline phosphatase (AP) staining for T cell subsets (CD4 and CD8) of smears from fingerprick blood functioned well under tropical climatic conditions when smears were stored frozen with silica gel before being labelled. Unlabelled smears were stored for up to 12 months and could...

  16. TRANSPARENT COATINGS FOR SOLAR CELLS RESEARCH

    Energy Technology Data Exchange (ETDEWEB)

    Glatkowski, P. J.; Landis, D. A.

    2013-04-16

    Todays solar cells are fabricated using metal oxide based transparent conductive coatings (TCC) or metal wires with optoelectronic performance exceeding that currently possible with Carbon Nanotube (CNT) based TCCs. The motivation for replacing current TCC is their inherent brittleness, high deposition cost, and high deposition temperatures; leading to reduced performance on thin substrates. With improved processing, application and characterization techniques Nanofiber and/or CNT based TCCs can overcome these shortcomings while offering the ability to be applied in atmospheric conditions using low cost coating processes At todays level of development, CNT based TCC are nearing commercial use in touch screens, some types of information displays (i.e. electronic paper), and certain military applications. However, the resistivity and transparency requirements for use in current commercial solar cells are more stringent than in many of these applications. Therefore, significant research on fundamental nanotube composition, dispersion and deposition are required to reach the required performance commanded by photovoltaic devices. The objective of this project was to research and develop transparent conductive coatings based on novel nanomaterial composite coatings, which comprise nanotubes, nanofibers, and other nanostructured materials along with binder materials. One objective was to show that these new nanomaterials perform at an electrical resistivity and optical transparency suitable for use in solar cells and other energy-related applications. A second objective was to generate new structures and chemistries with improved resistivity and transparency performance. The materials also included the binders and surface treatments that facilitate the utility of the electrically conductive portion of these composites in solar photovoltaic devices. Performance enhancement venues included: CNT purification and metallic tube separation techniques, chemical doping, CNT

  17. Proteomic response to 5,6-dimethylxanthenone 4-acetic acid (DMXAA, vadimezan in human non-small cell lung cancer A549 cells determined by the stable-isotope labeling by amino acids in cell culture (SILAC approach

    Directory of Open Access Journals (Sweden)

    Pan ST

    2015-02-01

    Full Text Available Shu-Ting Pan,1,* Zhi-Wei Zhou,2,3,* Zhi-Xu He,3 Xueji Zhang,4 Tianxin Yang,5 Yin-Xue Yang,6 Dong Wang,7 Jia-Xuan Qiu,1 Shu-Feng Zhou2 1Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 3Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, 4Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China; 5Department of Internal Medicine, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, UT, USA; 6Department of Colorectal Surgery, General Hospital of Ningxia Medical University, Yinchuan, 7Cancer Center, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing, People’s Republic of China *These two authors contributed equally to this work Abstract: 5,6-Dimethylxanthenone 4-acetic acid (DMXAA, also known as ASA404 and vadimezan, is a potent tumor blood vessel-disrupting agent and cytokine inducer used alone or in combination with other cytotoxic agents for the treatment of non-small cell lung cancer (NSCLC and other cancers. However, the latest Phase III clinical trial has shown frustrating outcomes in the treatment of NSCLC, since the therapeutic targets and underlying mechanism for the anticancer effect of DMXAA are not yet fully understood. This study aimed to examine the proteomic response to DMXAA and unveil the global molecular targets and possible mechanisms for the anticancer effect of DMXAA in NSCLC A549 cells using a stable-isotope labeling by amino acids in cell culture (SILAC approach. The proteomic data showed that treatment with DMXAA

  18. Labelling of Cells Engaged in DNA Synthesis: Autoradiography and BrdU Staining

    DEFF Research Database (Denmark)

    Madsen, Peder Søndergaard

    2010-01-01

    The cell cycle is divided in four phases: G1 phase, S phase (DNA-synthesis), G2 phase (together termed interphase) and M phase (mitosis). Cells that have ceased proliferation enter a state of quiescence called G0. M phase is itself composed of two tightly coupled processes: mitosis, in which...

  19. Monitoring virus entry into living cells using DiD-labeled dengue virus particles

    NARCIS (Netherlands)

    Ayala Nunez, Vanesa; Wilschut, Jan; Smit, Jolanda M.

    2011-01-01

    A variety of approaches can be applied to investigate the multiple steps and interactions that occur during virus entry into the host cell. Single-virus tracking is a powerful real-time imaging technique that offers the possibility to monitor virus-cell binding, internalization, intracellular traffi

  20. THE ENHANCED GREEN FLUORESCENT PROTEIN AS A MARKER FOR HUMAN TUMOR CELLS LABELLED BY RETROVIRAL TRANSDUCTION

    Institute of Scientific and Technical Information of China (English)

    傅建新; 王玮; 白霞; 卢大儒; 阮长耿; 陈子兴

    2002-01-01

    Objective: To investigate the feasibility of marking the human tumor cells with enhanced green fluorescent protein (EGFP) in vitro. Methods: The retroviral vector LGSN encoding EGFP was constructed and three human tumor cell lines were infected with LGSN amphotropic virus. Tumor cell lines that stably express EGFP were selected with G418. The integration and expression of EGFP gene were analyzed by polymerase chain reaction, and flow cytometry (FCM). Results: After gene transfection and ping-pong transduction, amphotropic producer line Am12/LGSN was generated with a stable green fluorescence signal readily detectable by FCM in up to 97% of examined cells. The viral titer in the supernatants was up to 8.2×105CFU/ml. After transduction and selection, G418-resistant leukemia K562, mammary carcinoma MCF-7, and bladder cancer 5637 cells were developed, in which the integration of both EGFP and neomycin resistance gene was confirmed by DNA amplification. In comparison with uninfected cells, FCM analysis revealed EGFP expression in up to 90% (range 85.5%~90.0%) of tumor cells containing LGSN provirus. Conclusion: The retroviral vector LGSN can effectively mark the human tumor cells with a stably EGFP expression which may be in studying tumor growth, metastasis and angiogenesis.

  1. Splenic sequestration of Tc-99m labeled heat treated red blood cells. [Feasibility of use for spleen imaging

    Energy Technology Data Exchange (ETDEWEB)

    Atkins, H L; Goldman, A G; Fairchild, R G; Oster, Z H; Som, P; Richards, P; Meinken, G E; Srivastava, S C

    1979-01-01

    The rate of blood clearance and spleen uptake as well as the total spleen uptake of heat damaged red blood cells labeled with Tc-99m was determined in eight patients, six of whom had chronic lymphatic leukemia, one had polycythemia vera and one had eosinophilia of unknown origin. Spleen uptake at 2 hrs was 72.0 +- 18.5%. Approximately 82.6% of the initial radioactivity was cleared from the blood by 2 hrs with a rapid T 1/2 component of 6.3 +- 4.7 min. The T 1/2 of splenic uptake was 8.3 +- 4.6 min with a plateauing of splenic radioactivity by 30 min. The preliminary results indicate that the method of preparation is reliable but the usefulness of the method for evaluating spleen function remains to be determined.

  2. Flow Cytometric DNA Analysis Using Cytokeratin Labeling for Identification of Tumor Cells in Carcinomas of the Breast and the Female Genital Tract

    Directory of Open Access Journals (Sweden)

    Rainer Kimmig

    2001-01-01

    Full Text Available Flow cytometric assessment of DNA‐ploidy and S‐phase fraction in malignant tumors is compromised by the heterogeneity of cell subpopulations derived from the malignant and surrounding connective tissue, e.g., tumor, stromal and inflammatory cells. To evaluate the effect on quality of DNA cell cycle analysis and determination of DNA ploidy, cytokeratin labeling of epithelial cells was used for tumor cell enrichment in breast, ovarian, cervical and endometrial cancer prior to DNA analysis. In a prospective study, tumor cell subpopulations of 620 malignant tumors were labeled by a FITC‐conjugated cytokeratin antibody (CK 5, 6, CK18 and CK 5, 6, 8 and CK 17, respectively prior to flow cytometric cell cycle analysis. Compared to total cell analysis, detection rate of DNA‐aneuploid tumors following cytokeratin labeling was increased from 62% to 76.5% in breast cancer, from 68% to 77% in ovarian cancer, from 60% to 80% in cervical cancer and from 30% to 53% in endometrial cancer. Predominantly in DNA‐diploid tumors, a significantly improved detection of S‐phase fraction of the tumor cells was shown due to the elimination of contaminating nonproliferating “normal cells”. S‐phase fraction following tumor cell enrichment was increased by 10% (mean following cytokeratin staining in ovarian and endometrial cancer, by 30% in breast cancer and even by 70% in cervical cancer compared to total cell analysis. Thus, diagnostic accuracy of DNA‐analysis was enhanced by cytokeratin labeling of tumor cells for all tumor entities investigated.

  3. Completion of proteomic data sets by Kd measurement using cell-free synthesis of site-specifically labeled proteins.

    Directory of Open Access Journals (Sweden)

    Paul Majkut

    Full Text Available The characterization of phosphotyrosine mediated protein-protein interactions is vital for the interpretation of downstream pathways of transmembrane signaling processes. Currently however, there is a gap between the initial identification and characterization of cellular binding events by proteomic methods and the in vitro generation of quantitative binding information in the form of equilibrium rate constants (Kd values. In this work we present a systematic, accelerated and simplified approach to fill this gap: using cell-free protein synthesis with site-specific labeling for pull-down and microscale thermophoresis (MST we were able to validate interactions and to establish a binding hierarchy based on Kd values as a completion of existing proteomic data sets. As a model system we analyzed SH2-mediated interactions of the human T-cell phosphoprotein ADAP. Putative SH2 domain-containing binding partners were synthesized from a cDNA library using Expression-PCR with site-specific biotinylation in order to analyze their interaction with fluorescently labeled and in vitro phosphorylated ADAP by pull-down. On the basis of the pull-down results, selected SH2's were subjected to MST to determine Kd values. In particular, we could identify an unexpectedly strong binding of ADAP to the previously found binding partner Rasa1 of about 100 nM, while no evidence of interaction was found for the also predicted SH2D1A. Moreover, Kd values between ADAP and its known binding partners SLP-76 and Fyn were determined. Next to expanding data on ADAP suggesting promising candidates for further analysis in vivo, this work marks the first Kd values for phosphotyrosine/SH2 interactions on a phosphoprotein level.

  4. Nutrition Labeling

    DEFF Research Database (Denmark)

    Grunert, Klaus G

    2013-01-01

    because consumers will avoid products that the label shows to be nutritionally deficient, but also because food producers will try to avoid marketing products that appear, according to the label, as nutritionally problematic, for example, because of a high content of saturated fat or salt. Nutrition......Nutrition labeling refers to the provision of information on a food product’s nutritional content on the package label. It can serve both public health and commercial purposes. From a public health perspective, the aim of nutrition labeling is to provide information that can enable consumers...... to make healthier choices when choosing food products. Nutrition labeling is thus closely linked to the notion of the informed consumer, that chooses products according to their aims, on the basis of the information at their disposal. Because many consumers are assumed to be interested in making healthy...

  5. Studies of the intermediary metabolism in cultured cells of the insect Spodoptera frugiperda using 13C- or 15N-labelled tracers

    Directory of Open Access Journals (Sweden)

    Bacher Adelbert

    2005-11-01

    Full Text Available Abstract Background Insect cells can serve as host systems for the recombinant expression of eukaryotic proteins. Using this platform, the controlled expression of 15N/13C labelled proteins requires the analysis of incorporation paths and rates of isotope-labelled precursors present in the medium into amino acids. For this purpose, Spodoptera frugiperda cells were grown in a complex medium containing [U-13C6]glucose. In a second experiment, cultures of S. frugiperda were grown in the presence of 15N-phenylalanine. Results Quantitative NMR analysis showed incorporation of the proffered [U-13C6]glucose into the ribose moiety of ribonucleosides (40 – 45% and into the amino acids, alanine (41%, glutamic acid/glutamine (C-4 and C-5, 30% and aspartate/asparagine (15%. Other amino acids and the purine ring of nucleosides were not formed from exogenous glucose in significant amounts (> 5%. Prior to the incorporation into protein the proffered 15N-phenylalanine lost about 70% of its label by transamination and the labelled compound was not converted into tyrosine to a significant extent. Conclusion Growth of S. frugiperda cells in the presence of [U-13C6]glucose is conducive to the fractional labelling of ribonucleosides, alanine, glutamic acid/glutamine and aspartic acid/asparagine. The isotopolog compositions of the ribonucleosides and of alanine indicate considerable recycling of carbohydrate intermediates in the reductive branch of the pentose phosphate pathway. The incorporation of 15N-labelled amino acids may be hampered by loss of the 15N-label by transamination.

  6. Endothelialization of Magnetic Graft Materials using SPION-labeled Endothelial Cells

    Science.gov (United States)

    Newman, Brant R.; Dragomir-Daescu, Dan; Harbuzariu, Adriana; McIntosh, Malcolm; Harburn, J. Jonathan; Parakka, Anthony; Kalra, Manju; Holmes, David; Simari, Robert D.; Sandhu, Gurpreet S.

    2010-12-01

    Seeding vascular grafts with autologous endothelial cells (EC) has been shown to improve in vivo patency, but high cost and development time have prevented widespread clinical use. A technique for loading EC with superparamagnetic iron-oxide nanospheres (SPIONs) was recently described. SPION-loaded EC experience magnetic attractive forces in the presence of sufficient magnetic field gradients. Using a multi-factorial design of experiments approach, the quantity and spatial distribution of magnetizable metal particles within a poly (ether urethane) matrix were systematically varied to produce unique material specimens. Specimens were seeded with SPION-loaded ECs, and cell coverage was quantified at various post-seeding time intervals using micrographic image analysis. The effects of changing design parameters on cell capture and sustained cell viability on magnetic substrates were statistically examined. Magnetized ferrites and samarium cobalt demonstrated cell capture, though cytotoxicity prevented sustained cell growth. Cobalt chromium substrates showed effective cell capture and growth to near complete confluence for up to one month.

  7. Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

    Energy Technology Data Exchange (ETDEWEB)

    Horita, Nobukatsu [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Tsuchiya, Kiichiro, E-mail: kii.gast@tmd.ac.jp [Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University (Japan); Hayashi, Ryohei [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Department of Gastroenterology and Metabolism, Hiroshima University (Japan); Fukushima, Keita; Hibiya, Shuji; Fukuda, Masayoshi; Kano, Yoshihito; Mizutani, Tomohiro; Nemoto, Yasuhiro; Yui, Shiro [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Okamoto, Ryuichi; Nakamura, Tetsuya [Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University (Japan); Watanabe, Mamoru [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan)

    2014-11-28

    Highlights: • Lentivirus mixed with Matrigel enables direct infection of intestinal organoids. • Our original approach allows the marking of a single stem cell in a crypt. • Time-lapse imaging shows the dynamics of a single stem cell. • Our lentivirus transgene system demonstrates plural long-lived stem cells in a crypt. - Abstract: Background and aims: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. Methods: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. Results: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. Conclusions: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.

  8. Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

    International Nuclear Information System (INIS)

    Highlights: • Lentivirus mixed with Matrigel enables direct infection of intestinal organoids. • Our original approach allows the marking of a single stem cell in a crypt. • Time-lapse imaging shows the dynamics of a single stem cell. • Our lentivirus transgene system demonstrates plural long-lived stem cells in a crypt. - Abstract: Background and aims: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. Methods: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. Results: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. Conclusions: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus

  9. Tumour cell recruitment of the JB-1 and L 1210 ascites tumour determined directly by double labelling with [14C]- and [3H]-thymidine.

    Science.gov (United States)

    Maurer-Schultze, B; Kondziella, U; Böswald, M

    1988-07-01

    Tumour cell recruitment of the JB-1 and L 1210 ascites tumour has been demonstrated directly by a double-labelling method with [14C]- and [3H]-thymidine (TdR). After [14C]-labelling of all proliferating tumour cells by multiple injections of [14C]TdR, recruitment of resting cells was stimulated by removal of the majority of tumour cells, i.e. by maximum aspiration of ascitic fluid. The number of recruited resting cells in the remaining tumour that re-enter the cell cycle after stimulation was demonstrated directly by a single injection of [3H]TdR given at different times after stimulation. The increase in the percentage of purely [3H]-labelled cells, i.e. recruited cells, with increasing time after stimulation, shows that recruitment is not a synchronous but a continuous process, the maximum of which occurs earlier in the case of the L 1210 than the JB-1 tumour. This suggests that there seems to be a relationship between the time required for maximum recruitment and the corresponding cell cycle parameters of the unperturbed tumour. There is a transitory increase of the growth fraction to about 100% and a considerable shortening of the cycle time at the maximum of recruitment.

  10. Changes in diacylglycerol labeling, cell shape, and protein phosphorylation distinguish triggering from activation of human neutrophils

    International Nuclear Information System (INIS)

    Upon activation neutrophils release reactive oxygen intermediates such as superoxide anion (O2-) which are potent mediators of inflammation. Various agents elicit different responses. In contrast, phorbol myristate acetate (PMA, 1.6 μM) acting directly via protein kinase C is a potent stimulus for O2-. The authors compared the kinetics of appearance of various second messengers with the capacity of these ligands to elicit O2- generation. Kinetic analysis showed a two-phase response to membrane ligands; both an early (≥ 15 s) and a late (>15 s) increase in [3H]- and [14C]diacylglycerol (DG) was noted in response to fMLP. In contrast, LTB4 elicited only a rapid early increase in DG. The rise in DG evoked by PMA was late. Moreover, comparison of increases in [3H]DG versus those of [14C]DG at early and late time points suggested that DG was not formed exclusively from the hydrolysis of polyphosphoinositides. Kinetic analysis of protein phosphorylation was compared to the early and late increments of DG labeling. A 47,000 M/sub r/ protein was phosphorylated with kinetics consistent with the production of O2- and DG in response to fMLP and PMA. The temporal pattern of the formation of diacylglycerol and the phosphorylation of proteins describe a dual signal. The data suggest that neutrophils require not only triggering (the rapid generation of a signal) but also activation (the maintenance of a signal) to sustain responses

  11. A New Method for Preparing Mesenchymal Stem Cells and Labeling with Ferumoxytol for Cell Tracking by MRI

    OpenAIRE

    Li Liu; Lanya Tseng; Qing Ye; Wu, Yijen L.; Bain, Daniel J.; Chien Ho

    2016-01-01

    Mesenchymal stem cells (MSCs) are among the major stem cells used for cell therapy and regenerative medicine. In-vivo cell-tracking by magnetic resonance imaging (MRI) is crucial for regenerative medicine, allowing verification that the transplanted cells reach the targeted sites. Cellular MRI combined with superparamagnetic iron-oxide (SPIO) contrast agents is an effective cell-tracking method. Here, we are reporting a new “bio-mimicry” method by making use of the “in-vivo environment” of MS...

  12. Immunocytochemical labelling of aerobic and hypoxic mammalian cells using a platinated derivative of EF5.

    OpenAIRE

    Matthews, J.; Adomat, H.; Farrell, N; King, P.; Koch, C.; Lord, E.; Palcic, B.; Poulin, N; Sangulin, J.; Skov, K.

    1996-01-01

    The monoclonal antibody ELK3-51 was previously developed to detect adducts of the 2-nitroimidazole EF5. Direct immunofluorescence was used to detect adducts of EF5 or of a platinated derivative cis-[PtCl2(NH3)EF5] in SCCVII cells treated under aerobic or hypoxic conditions. Fluorescence measurements of these cells using both image and flow cytometric methods were compared, giving similar profiles. Platination significantly decreased immunofluorescence levels (approximately 4-fold less than EF...

  13. Identification of secreted proteins regulated by cAMP in glioblastoma cells using glycopeptide capture and label-free quantification.

    Science.gov (United States)

    Hill, Jennifer J; Moreno, Maria J; Lam, Jean C Y; Haqqani, Arsalan S; Kelly, John F

    2009-02-01

    Exposure of glioblastoma U87MG cells to a cAMP analog leads to a decrease in proliferation, invasion, and angiogenic potential. Here, we apply a label-free MS-based approach to identify formerly N-linked glycopeptides that change in abundance upon cAMP treatment. Over 150 unique glycopeptides in three biological repetitions were quantified, leading to the identification of 14 upregulated proteins and 21 downregulated proteins due to cAMP treatment. Of these, eight have been validated, either through comparison with microarray data or by Western blot. We estimate our ability to identify differentially expressed peptides at greater than 85% in a single biological repetition, while the analysis of multiple biological repetitions lowers the false positive rate to approximately 2%. Many of the proteins identified in this study are involved in cell signaling and some, such as Tenascin C, Cathepsin L, Neuroblastoma suppressor of tumorigenicity, and AXL/UFO tyrosine-protein kinase receptor, have been previously shown to be involved in glioblastoma progression. We also identify several semitryptic peptides that increase in abundance upon cAMP treatment, suggesting that cAMP regulates protease activity in these cells. Overall, these results demonstrate the benefits of using a highly specific enrichment method for quantitative proteomic experiments. PMID:19137551

  14. Dataset of differential lipid raft and global proteomes of SILAC-labeled cystic fibrosis cells upon TNF -α stimulation.

    Science.gov (United States)

    Chhuon, C; Pranke, I; Borot, F; Tondelier, D; Lipecka, J; Fritsch, J; Chanson, M; Edelman, A; Ollero, M; Guerrera, I C

    2016-12-01

    Cystic fibrosis (CF) is a genetic disease due to mutations in the cystic fibrosis transmembrane regulator (CFTR), F508del-CFTR being the most frequent. Lipid raft-like microdomains (LRM) are regions of the plasma membrane that present a high cholesterol content and are insoluble to non-ionic detergents. LRM are essential functional and structural platforms that play an important role in the inflammatory response. CFTR is a known modulator of inflammation in LRM. Here we provide mass spectrometry data on the global impact of CFTR mutation and TNF-a stimulation on the LRM proteome. We used the Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) approach to quantify and identify 332 proteins in LRM upon TNF-a stimulation in CF cells and 1381 for the global proteome. We report two detailed tables containing lists of proteins obtained by mass spectrometry and the immunofluorescence validation results for one of these proteins, the G-protein coupled receptor 5A. These results are associated with the article "Changes in lipid raft proteome upon TNF-α stimulation of cystic fibrosis cells" (Chhuon et al., in press [1]).

  15. Label-free separation of human embryonic stem cells and their differentiating progenies by phasor fluorescence lifetime microscopy

    Science.gov (United States)

    Stringari, Chiara; Sierra, Robert; Donovan, Peter J.; Gratton, Enrico

    2012-04-01

    We develop a label-free optical technique to image and discriminate undifferentiated human embryonic stem cells (hESCs) from their differentiating progenies in vitro. Using intrinsic cellular fluorophores, we perform fluorescence lifetime microscopy (FLIM) and phasor analysis to obtain hESC metabolic signatures. We identify two optical biomarkers to define the differentiation status of hESCs: Nicotinamide adenine dinucleotide (NADH) and lipid droplet-associated granules (LDAGs). These granules have a unique lifetime signature and could be formed by the interaction of reactive oxygen species and unsaturated metabolic precursor that are known to be abundant in hESC. Changes in the relative concentrations of these two intrinsic biomarkers allow for the discrimination of undifferentiated hESCs from differentiating hESCs. During early hESC differentiation we show that NADH concentrations increase, while the concentration of LDAGs decrease. These results are in agreement with a decrease in oxidative phosphorylation rate. Single-cell phasor FLIM signatures reveal an increased heterogeneity in the metabolic states of differentiating H9 and H1 hESC colonies. This technique is a promising noninvasive tool to monitor hESC metabolism during differentiation, which can have applications in high throughput analysis, drug screening, functional metabolomics and induced pluripotent stem cell generation.

  16. Fluorescently labeled methyl-beta-cyclodextrin enters intestinal epithelial Caco-2 cells by fluid-phase endocytosis.

    Directory of Open Access Journals (Sweden)

    Ferenc Fenyvesi

    Full Text Available Cyclodextrins are widely used excipients for increasing the bioavailability of poorly water-soluble drugs. Their effect on drug absorption in the gastrointestinal tract is explained by their solubility- and permeability-enhancement. The aims of this study were to investigate penetration properties of fluorescently labeled randomly methylated-beta-cyclodextrin (FITC-RAMEB on Caco-2 cell layer and examine the cellular entry of cyclodextrins on intestinal cells. The permeability of FITC-RAMEB through Caco-2 monolayers was very limited. Using this compound in 0.05 mM concentration the permeability coefficient was 3.35±1.29×10(-8 cm/s and its permeability did not change in the presence of 5 mM randomly methylated-beta-cyclodextrin. Despite of the low permeability, cellular accumulation of FITC-RAMEB in cytoplasmic vesicles was significant and showed strong time and concentration dependence, similar to the characteristics of the macropinocytosis marker Lucifer Yellow. The internalization process was fully inhibited at 0°C and it was drastically reduced at 37°C applying rottlerin, an inhibitor of macropinocytosis. Notably, FITC-RAMEB colocalized with the early endosome organizer Rab5a. These results have revealed that FITC-RAMEB is able to enter intestinal epithelial cells by fluid-phase endocytosis from the apical side. This mechanism can be an additional process which helps to overcome the intestinal barrier and contributes to the bioavailability enhancement of cyclodextrins.

  17. Effect of an extract of Artemisia vulgaris L. (Mugwort) on the in vitro labeling of red blood cells and plasma proteins with technetium-99m

    International Nuclear Information System (INIS)

    The aim of this work was to evaluate the effect of an extract of the Artemisia vulgaris L. (mugwort) on the labeling of blood constituents with technetium-99m (99mTc). Blood samples from Wistar rats were incubated with a mugwort extract and the radiolabeling of blood constituents was carried out. Plasma and blood cells were separated by centrifugation. Aliquots of plasma and blood cells were also precipitated with trichloroacetic acid and centrifuged to isolate soluble and insoluble fractions of plasma and blood cells. Radioactivity in each fraction was counted and the percentages of radioactivity (%ATI) was calculated. Mugwort extract decreased significantly (p<0.05) the %ATI on the blood compartments and on the blood cells proteins (insoluble fraction). The analysis of the results indicates that the extract could have substances that could interfere on the transport of stannous through the erythrocyte membrane altering the labeling of blood cells with 99mTc. (author)

  18. Isolation and In-111-Oxine Labeling of Murine NK Cells for Assessment of Cell Trafficking in Orthotopic Lung Tumor Model

    NARCIS (Netherlands)

    Malviya, Gaurav; Nayak, Tapan; Gerdes, Christian; Dierckx, Rudi A. J. O.; Signore, Alberto; de Vries, Erik F. J.

    2016-01-01

    A noninvasive in vivo imaging method for NK cell trafficking is essential to gain further understanding of the pathogenesis of NK cell mediated immune response to the novel cancer treatment strategies, and to discover the homing sites and physiological distribution of NK cells. Although human NK tel

  19. Applications and technical challenges of fluorescence in situhybridization in stem cell research

    Energy Technology Data Exchange (ETDEWEB)

    Weier, Heinz-Ulli G.; Chu, Lisa W.; Murnane, John P.; Weier,Jingly F.

    2003-07-02

    Stem cell research, maintenance and manipulations have advanced significantly in recent years, and we now witness successful clinical applications of stem therapies. However, challenges in regard to karyotypic stability and the ploidy status of stem cell lines have been addressed only marginally. Our approach to develop technology to address these highly relevant issues is based on fluorescence in situ hybridization (FISH) using non-isotopically labeled DNA probes. As a single cell analysis technique, FISH is expected to be applicable to a variety of cells and tissues including interphase and metaphase cell preparations as well as tissue sections and biopsy material. Over the last decade, our laboratories generated a large number of probes and probe sets for the molecular cytogenetic analyses of stem cells derived from different species. These probes and the introduction of Spectral Imaging bring us close to be able to perform a comprehensive karyotype analysis of single interphase cell nuclei. It should furthermore be possible to couple cytogenetic investigations of the cellular genotype with analysis of gene expression. This report summarizes our technical achievements relevant to stem cell research, and outlines plans for future research and developments.

  20. Immunocytochemical labelling of aerobic and hypoxic mammalian cells using a platinated derivative of EF5.

    Science.gov (United States)

    Matthews, J; Adomat, H; Farrell, N; King, P; Koch, C; Lord, E; Palcic, B; Poulin, N; Sangulin, J; Skov, K

    1996-07-01

    The monoclonal antibody ELK3-51 was previously developed to detect adducts of the 2-nitroimidazole EF5. Direct immunofluorescence was used to detect adducts of EF5 or of a platinated derivative cis-[PtCl2(NH3)EF5] in SCCVII cells treated under aerobic or hypoxic conditions. Fluorescence measurements of these cells using both image and flow cytometric methods were compared, giving similar profiles. Platination significantly decreased immunofluorescence levels (approximately 4-fold less than EF5) after 3 h in hypoxia, but also increased levels after exposure in air (approximately 1.5 x) such that the hypoxic ratio decreased from approximately 50 to approximately 13. Platinated EF5 also showed significantly greater cytotoxicity than its parent in both aerobic and hypoxic cells. These results are consistent with targeting of EF5 to DNA, which was confirmed qualitatively by confocal microscopy. PMID:8763880

  1. 76 FR 23599 - Findings of Research Misconduct

    Science.gov (United States)

    2011-04-27

    ... and switched the labels on four (4) cell culture dishes for cells used in the same type of experiments... tampered with laboratory research materials by adding ethanol to his colleague's cell culture media,...

  2. Differentiating intratumoral melanocytes from Langerhans cells in nonmelanocytic pigmented skin tumors in vivo by label-free third-harmonic generation microscopy

    Science.gov (United States)

    Weng, Wei-Hung; Liao, Yi-Hua; Tsai, Ming-Rung; Wei, Ming-Liang; Huang, Hsin-Yi; Sun, Chi-Kuang

    2016-07-01

    Morphology and distribution of melanocytes are critical imaging information for the diagnosis of melanocytic lesions. However, how to image intratumoral melanocytes noninvasively in pigmented skin tumors is seldom investigated. Third-harmonic generation (THG) is shown to be enhanced by melanin, whereas high accuracy has been demonstrated using THG microscopy for in vivo differential diagnosis of nonmelanocytic pigmented skin tumors. It is thus desirable to investigate if label-free THG microscopy was capable to in vivo identify intratumoral melanocytes. In this study, histopathological correlations of label-free THG images with the immunohistochemical images stained with human melanoma black (HMB)-45 and cluster of differentiation 1a (CD1a) were made. The correlation results indicated that the intratumoral THG-bright dendritic-cell-like signals were endogenously derived from melanocytes rather than Langerhans cells (LCs). The consistency between THG-bright dendritic-cell-like signals and HMB-45 melanocyte staining showed a kappa coefficient of 0.807, 84.6% sensitivity, and 95% specificity. In contrast, a kappa coefficient of -0.37, 21.7% sensitivity, and 30% specificity were noted between the THG-bright dendritic-cell-like signals and CD1a staining for LCs. Our study indicates the capability of noninvasive label-free THG microscopy to differentiate intratumoral melanocytes from LCs, which is not feasible in previous in vivo label-free clinical-imaging modalities.

  3. In vitro studies using Tc-99m-labelled human serum albumin millimicrospheres for assessing the contribution to phagocytosis of mononuclear blood cells

    International Nuclear Information System (INIS)

    Tc-99m-labelled HSA millimicrospheres were incubated with whole blood for various periods of time, in order to analyse the rate of fixation of HSA millimicrospheres to monocytes, and their interaction. It was not possible with the technique applied to differentiate between a genuine process of phagocytosis and a conceivable mere fixation of particles to the cells. (MBC)

  4. TRANSPARENT COATINGS FOR SOLAR CELLS RESEARCH

    Energy Technology Data Exchange (ETDEWEB)

    Glatkowski, P.J.; Landis, D.A.

    2013-04-16

    Todays solar cells are fabricated using metal oxide based transparent conductive coatings (TCC) or metal wires with optoelectronic performance exceeding that currently possible with Carbon Nanotube (CNT) based TCCs. The motivation for replacing current TCC is their inherent brittleness, high deposition cost, and high deposition temperatures; leading to reduced performance on thin substrates. With improved processing, application and characterization techniques Nanofiber and/or CNT based TCCs can overcome these shortcomings while offering the ability to be applied in atmospheric conditions using low cost coating processes At todays level of development, CNT based TCC are nearing commercial use in touch screens, some types of information displays (i.e. electronic paper), and certain military applications. However, the resistivity and transparency requirements for use in current commercial solar cells are more stringent than in many of these applications. Therefore, significant research on fundamental nanotube composition, dispersion and deposition are required to reach the required performance commanded by photovoltaic devices. The objective of this project was to research and develop transparent conductive coatings based on novel nanomaterial composite coatings, which comprise nanotubes, nanofibers, and other nanostructured materials along with binder materials. One objective was to show that these new nanomaterials perform at an electrical resistivity and optical transparency suitable for use in solar cells and other energy-related applications. A second objective was to generate new structures and chemistries with improved resistivity and transparency performance. The materials also included the binders and surface treatments that facilitate the utility of the electrically conductive portion of these composites in solar photovoltaic devices. Performance enhancement venues included: CNT purification and metallic tube separation techniques, chemical doping, CNT

  5. Ground Zero in the Debate over Stem-Cell Research.

    Science.gov (United States)

    Southwick, Ron

    2001-01-01

    Describes how political, legal, and ethical battles over embryonic stem-cell research are focused on the University of Wisconsin at Madison, where the cells were first isolated. Addresses the issue of access to the university's stem cells and a recent presidential decision regarding funding for stem-cell research.(EV)

  6. Specific labeling of peptidoglycan precursors as a tool for bacterial cell wall studies

    NARCIS (Netherlands)

    van Dam, V.; Olrichs, N.K.; Breukink, E.J.

    2009-01-01

    Wall chart: The predominant component of the bacterial cell wall, peptidoglycan, consists of long alternating stretches of aminosugar subunits interlinked in a large three-dimensional network and is formed from precursors through several cytosolic and membrane-bound steps. The high tolerance of the

  7. A fluorescent and chemiluminescent difunctional mesoporous silica nanoparticle as a label for the ultrasensitive detection of cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Tao Liang [Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi' an 710062 (China); Song Chaojun; Sun Yuanjie [Department of Immunology, The Fourth Military Medical University, Xi' an 710032 (China); Li Xiaohua; Li Yunyun [Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi' an 710062 (China); Jin Boquan [Department of Immunology, The Fourth Military Medical University, Xi' an 710032 (China); Zhang Zhujun, E-mail: zhangzj@snnu.edu.cn [Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi' an 710062 (China); Yang Kun, E-mail: yangkunkun@fmmu.edu