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Sample records for fepro cell labeling

  1. Effects of Ferumoxides – Protamine Sulfate Labeling on Immunomodulatory Characteristics of Macrophage-like THP-1 Cells

    PubMed Central

    Janic, Branislava; Iskander, A. S. M.; Rad, Ali M.; Soltanian-Zadeh, Hamid; Arbab, Ali S.

    2008-01-01

    Superparamagnetic Iron Oxide (SPIO) complexed with cationic transfection agent is used to label various mammalian cells. Labeled cells can then be utilized as an in vivo magnetic resonance imaging (MRI) probes. However, certain number of in vivo administered labeled cells may be cleared from tissues by the host's macrophages. For successful translation to routine clinical application of SPIO labeling method it is important that this mode of in vivo clearance of iron does not elicit any diverse immunological effects. The purpose of this study was to demonstrate that SPIO agent ferumoxides-protamine sulfate (FePro) incorporation into macrophages does not alter immunological properties of these cells with regard to differentiation, chemotaxis, and ability to respond to the activation stimuli and to modulate T cell response. We used THP-1 cell line as a model for studying macrophage cell type. THP-1 cells were magnetically labeled with FePro, differentiated with 100 nM of phorbol ester, 12-Myristate-13-acetate (TPA) and stimulated with 100 ng/ml of LPS. The results showed 1) FePro labeling had no effect on the changes in morphology and expression of cell surface proteins associated with TPA induced differentiation; 2) FePro labeled cells responded to LPS with slightly higher levels of NFκB pathway activation, as shown by immunobloting; TNF-α secretion and cell surface expression levels of CD54 and CD83 activation markers, under these conditions, were still comparable to the levels observed in non-labeled cells; 3) FePro labeling exhibited differential, chemokine dependent, effect on THP-1 chemotaxis with a decrease in cell directional migration to MCP-1; 4) FePro labeling did not affect the ability of THP-1 cells to down-regulate T cell expression of CD4 and CD8 and to induce T cell proliferation. Our study demonstrated that intracellular incorporation of FePro complexes does not alter overall immunological properties of THP-1 cells. The described experiments provide

  2. Label-free cell profiling.

    PubMed

    Schasfoort, Richard B M; Bentlage, Arthur E H; Stojanovic, Ivan; van der Kooi, Alex; van der Schoot, Ellen; Terstappen, Leon W M M; Vidarsson, Gestur

    2013-08-01

    A surface plasmon resonance (SPR) array imaging method is outlined for label-free cell profiling. Red blood cells (RBCs) were injected into a flow chamber on top of a spotted sensor surface. Spots contained antibodies to various RBC membrane antigens. A typical sensorgram showed an initial response corresponding to cell sedimentation (S) followed by a specific upward response (T) corresponding to specific binding of cells during a critical wash step. The full analysis cycle for RBC profiling was less than 6 min. The sensor surface could be regenerated at least 100 times, allowing the determination of a cell surface antigen profile of RBCs.

  3. Stem cell labeling for magnetic resonance imaging.

    PubMed

    Himmelreich, Uwe; Hoehn, Mathias

    2008-01-01

    In vivo applications of cells for the monitoring of their cell dynamics increasingly use non-invasive magnetic resonance imaging. This imaging modality allows in particular to follow the migrational activity of stem cells intended for cell therapy strategies. All these approaches require the prior labeling of the cells under investigation for excellent contrast against the host tissue background in the imaging modality. The present review discusses the various routes of cell labeling and describes the potential to observe both cell localization and their cell-specific function in vivo. Possibilities for labeling strategies, pros and cons of various contrast agents are pointed out while potential ambiguities or problems of labeling strategies are emphasized.

  4. Recent developments in blood cell labeling research

    SciTech Connect

    Srivastava, S.C.; Straub, R.F.; Meinken, G.E.

    1988-09-07

    A number of recent developments in research on blood cell labeling techniques are presented. The discussion relates to three specific areas: (1) a new in vitro method for red blood cell labeling with /sup 99m/Tc; (2) a method for labeling leukocytes and platelets with /sup 99m/Tc; and (3) the use of monoclonal antibody technique for platelet labeling. The advantages and the pitfalls of these techniques are examined in the light of available mechanistic information. Problems that remain to be resolved are reviewed. An assessment is made of the progress as well as prospects in blood cell labeling methodology including that using the monoclonal antibody approach. 37 refs., 4 figs.

  5. The antibody approach of labeling blood cells

    SciTech Connect

    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.

  6. The antibody approach of labeling blood cells

    SciTech Connect

    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.

  7. The antibody approach of labeling blood cells

    SciTech Connect

    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. Detection and Quantification of Magnetically Labeled Cells by Cellular MRI

    PubMed Central

    Liu, Wei; Frank, Joseph A.

    2008-01-01

    Labeling cells with superparamagnetic iron oxide (SPIO) nanoparticles, paramagnetic contrast agent (gadolinium) or perfluorocarbons allows for the possibility of tracking single or clusters of labeled cells within target tissues following either direct implantation or intravenous injection. This review summarizes the practical issues regarding detection and quantification of magnetically labeled cells with various MRI contrast agents with a focus on SPIO nanoparticles. PMID:18995978

  9. Induced Pluripotent Stem Cell Labeling Using Quantum Dots

    PubMed Central

    Yukawa, Hiroshi; Suzuki, Kaoru; Kano, Yuki; Yamada, Tatsuya; Kaji, Noritada; Ishikawa, Tetsuya; Baba, Yoshinobu

    2013-01-01

    Induced pluripotent stem (iPS) cells have received remarkable attention as the cell sources for clinical applications of regenerative medicine including stem cell therapy. Additionally, labeling technology is in high demand for tracing transplanted cells used in stem cell therapy. In this study, we used quantum dots (QDs), which have distinct fluorescence abilities in comparison with traditional probes, as the labeling materials and investigated whether iPS cells could be labeled with QDs with no cytotoxicity. iPS cells could not be labeled with QDs alone but required the use of cell-penetrating peptides such as octaarginine (R8). No significant cytotoxicity to iPS cells was confirmed by up to 8 nM QDs, and the iPS cells labeled with QDs maintained their undifferentiated state and pluripotency. These data suggest that QDs can be used for fluorescence labeling of iPS cells. PMID:26858884

  10. Design of polymeric immunomicrospheres for cell labelling and cell separation

    NASA Technical Reports Server (NTRS)

    Rembaum, A.; Margel, S.

    1978-01-01

    Synthesis of several classes of hydrophylic microspheres applied to cell labeling and cell separation is described. Five classes of cross-linked microspheres with functional groups such as carboxyl, hydroxyl, amide and/or pyridine groups were synthesized. These functional groups were used to bind covalently antibodies and other proteins to the surface of the microspheres. To optimize the derivatisation technique, polyglutaraldehyde immunomicrospheres were prepared and utilized. Specific populations of human and murine lymphocytes were labelled with microspheres synthesized by the emulsion of the ionizing radiation technique. The labelling of the cells by means of microspheres containing an iron core produced successful separation of B from T lymphocytes by means of a magnetic field.

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

    PubMed

    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.

  12. Labeling of Mesenchymal Stem Cells by Bioconjugated Quantum Dots

    PubMed Central

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

    2015-01-01

    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. PMID:17887799

  13. Hemobilia detected by Tc-99m labeled red blood cells

    SciTech Connect

    Winzelberg, G.G.; Wholey, M.H.; Ismail-Beigi, F.

    1982-01-01

    Tc-99m labeled red blood cells were successfully used to determine the site of hemorrhage in a 67-year-old man with hemobilia. A false hepatic artery aneurysm was confirmed at angiography and ultimately successfully embolized. The relative merits of using Tc-99m labeled red blood cells for detecting sources of upper gastrointestinal bleeding are discussed.

  14. State of the science of blood cell labeling

    SciTech Connect

    Srivastava, S.C.; Straub, R.F.

    1989-01-01

    Blood cell labeling can be considered a science in as far as it is based on precise knowledge and can be readily reproduced. This benchmark criterion is applied to all current cell labeling modalities and their relative merits and deficiencies are discussed. Mechanisms are given where they are known as well as labeling yields, label stability, and cell functionality. The focus is on the methodology and its suitability to the clinical setting rather than on clinical applications per se. Clinical results are cited only as proof of efficacy of the various methods. The emphasis is on technetium as the cell label, although comparisons are made between technetium and indium, and all blood cells are covered. 52 refs., 6 figs., 7 tabs.

  15. Application of fluorescence labeled liposome nanoparticles in the cell imaging

    NASA Astrophysics Data System (ADS)

    Hu, Jianbing; Li, Huimin; He, Xiaoxiao; Gong, Ping; Wang, Kemin; Zhang, Shouchun

    2007-05-01

    Fluorescence labeled liposome nanoparticles were prepared by dispersion of film method. The size of nanoparticles was around 50 nm. DPPE-FITC synthesized in our lab was used to label the liposome nanoparticles. Anti-cytokeratins 19 antibody was connected to the surface of the fluorescence liposome nanoparticles. After incubation with MGC cells and COS-7 cells for 30 min, MGC cells were selectively recognized by anti-cytokeratins 19 antibody modified liposome nanoparticles and well imaged under laser confocal microscope. This fluorescence labeled liposome nanoparticles is expected to have good applications in cell recognition and tumor diagnosis.

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

    NASA Astrophysics Data System (ADS)

    Mo, Runyang; Yang, Jian; Wu, Ed X.; Lin, Shuyu

    2011-09-01

    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.

  17. Cell labeling with magnetic nanoparticles: Opportunity for magnetic cell imaging and cell manipulation

    PubMed Central

    2013-01-01

    This tutorial describes a method of controlled cell labeling with citrate-coated ultra small superparamagnetic iron oxide nanoparticles. This method may provide basically all kinds of cells with sufficient magnetization to allow cell detection by high-resolution magnetic resonance imaging (MRI) and to enable potential magnetic manipulation. In order to efficiently exploit labeled cells, quantify the magnetic load and deliver or follow-up magnetic cells, we herein describe the main requirements that should be applied during the labeling procedure. Moreover we present some recommendations for cell detection and quantification by MRI and detail magnetic guiding on some real-case studies in vitro and in vivo. PMID:24564857

  18. Carbon "Quantum" Dots for Fluorescence Labeling of Cells.

    PubMed

    Liu, Jia-Hui; Cao, Li; LeCroy, Gregory E; Wang, Ping; Meziani, Mohammed J; Dong, Yiyang; Liu, Yuanfang; Luo, Pengju G; Sun, Ya-Ping

    2015-09-01

    The specifically synthesized and selected carbon dots of relatively high fluorescence quantum yields were evaluated in their fluorescence labeling of cells. For the cancer cell lines, the cellular uptake of the carbon dots was generally efficient, resulting in the labeling of the cells with bright fluorescence emissions for both one- and two-photon excitations from predominantly the cell membrane and cytoplasm. In the exploration on labeling the live stem cells, the cellular uptake of the carbon dots was relatively less efficient, though fluorescence emissions could still be adequately detected in the labeled cells, with the emissions again predominantly from the cell membrane and cytoplasm. This combined with the observed more efficient internalization of the same carbon dots by the fixed stem cells might suggest some significant selectivity of the stem cells toward surface functionalities of the carbon dots. The needs and possible strategies for more systematic and comparative studies on the fluorescence labeling of different cells, including especially live stem cells, by carbon dots as a new class of brightly fluorescent probes are discussed.

  19. Labeling of lectin receptors during the cell cycle.

    PubMed

    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.

  20. Labeling of lectin receptors during the cell cycle.

    PubMed

    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

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

    NASA Astrophysics Data System (ADS)

    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.

  2. Stable isotope labeling of oligosaccharide cell surface antigens

    SciTech Connect

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

    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.

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

    PubMed

    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.

  4. X-ray microscopic studies of labeled nuclear cell structures

    NASA Astrophysics Data System (ADS)

    Vogt, S.; Schneider, G.; Steuernagel, A.; Lucchesi, J.; Schulze, E.; Rudolph, D.; Schmahl, G.

    2000-05-01

    In X-ray microscopy different proteins are not readily distinguishable. However, in cell biology it is often desirable to localize single proteins, e.g., inside the cell nucleus. This can be achieved by immunogold labeling. Colloidal gold conjugated antibodies are used to mark the protein specifically. With silver solution these are enlarged so as to heighten their contrast. The strong absorption of silver allows easy visualization of the label in the nuclei. In this study male specific lethal 1 protein in male Drosophila melanogaster cells was labeled. This protein forms, together with four other proteins, a complex that is associated with the male X chromosome. It regulates dosage compensation by enhancing X-linked gene transcription in males. Room temperature and cyro transmission X-ray microscopic images (taken with the Göttingen TXM at BESSY) of these labeled cells are shown. Confocal laser scan microscopy ascertains the correct identification of the label in the X-ray micrographs, and allows comparison of the structural information available from both instruments.

  5. Labeling cells for in vivo tracking using (19)F MRI.

    PubMed

    Srinivas, Mangala; Boehm-Sturm, Philipp; Figdor, Carl G; de Vries, I Jolanda; Hoehn, Mathias

    2012-12-01

    Noninvasive in vivo cell tracking is crucial to fully understand the function of mobile and/or transplanted cells, particularly immune cells and cellular therapeutics. (19)F MRI for cell tracking has several advantages; chief among them are its noninvasive nature which allows longitudinal data acquisition, use of a stable, non-radioactive isotope permitting long-term tracking, the absence of confounding endogenous signal, and the ability to quantify cell numbers from image data. However, generation of sufficient signal i.e. (19)F cell loading is a key challenge, particularly with non-phagocytic cells such as lymphocytes and stem cells. A range of (19)F cell labels have been developed, including emulsions, particles, polymers, and agents for clinical use. Various animal and primary human cells, such as dendritic cells, lymphocytes and phagocytes have been successfully labeled and studied in models of autoimmune disease, inflammation and transplant rejection. Primary human cells, particularly dendritic cells as used in vaccine therapy have been tested for imminent clinical application. Here, we summarize current cell loading strategies and sensitivity of in vivo cell imaging with (19)F MRI, and discuss the processing of image data for accurate quantification of cell numbers. This novel technology is uniquely applicable to the longitudinal and quantitative tracking of cells in vivo.

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

    PubMed

    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.

  7. Endothelial cell labeling with indium-111-oxine as a marker of cell attachment to bioprosthetic surfaces

    SciTech Connect

    Sharefkin, J.B.; Lather, C.; Smith, M.; Rich, N.M.

    1983-03-01

    Canine vascular endothelium labeled with indium-111-oxine was used as a marker of cell attachment to vascular prosthetic surfaces with complex textures. Primarily cultured and freshly harvested endothelial cells both took up the label rapidly. An average of 72% of a 32 micro Ci labeling dose was taken up by 1.5 X 10(6) cells in 10 min in serum-free medium. Over 95% of freshly labeled cells were viable by trypan blue tests and only 5% of the label was released after 1 h incubations at 37 degrees C. Labeled and unlabeled cells had similar rates of attachment to plastic dishes. Scanning electron microscopic studies showed that labeled cells retained their ability to spread on tissue culture dishes even at low (1%) serum levels. Labeled endothelial cells seeded onto Dacron or expanded polytetrafluoroethylene vascular prostheses by methods used in current surgical models could be identified by autoradiography of microscopic sections of the prostheses, and the efficiency of cell attachment to the prosthesis could be measured by gamma counting. Indium-111 labeling affords a simple and rapid way to measure initial cell attachment to, and distribution on, vascular prosthetic materials. The method could also allow measurement of early cell loss from a flow surface in vivo by using external gamma imaging.

  8. Antigen specific killing assay using CFSE labeled target cells.

    PubMed

    Durward, Marina; Harms, Jerome; Splitter, Gary

    2010-11-09

    Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

  9. Deep Learning in Label-free Cell Classification

    NASA Astrophysics Data System (ADS)

    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.

  10. Deep Learning in Label-free Cell Classification.

    PubMed

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

    2016-01-01

    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. PMID:26975219

  11. Deep Learning in Label-free Cell Classification.

    PubMed

    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.

  12. Deep Learning in Label-free Cell Classification

    PubMed Central

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

    2016-01-01

    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. PMID:26975219

  13. Deep Learning in Label-free Cell Classification

    DOE PAGESBeta

    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 individualmore » 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. In conclusion, this system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.« less

  14. Label-free density difference amplification-based cell sorting.

    PubMed

    Song, Jihwan; Song, Minsun; Kang, Taewook; Kim, Dongchoul; Lee, Luke P

    2014-11-01

    The selective cell separation is a critical step in fundamental life sciences, translational medicine, biotechnology, and energy harvesting. Conventional cell separation methods are fluorescent activated cell sorting and magnetic-activated cell sorting based on fluorescent probes and magnetic particles on cell surfaces. Label-free cell separation methods such as Raman-activated cell sorting, electro-physiologically activated cell sorting, dielectric-activated cell sorting, or inertial microfluidic cell sorting are, however, limited when separating cells of the same kind or cells with similar sizes and dielectric properties, as well as similar electrophysiological phenotypes. Here we report a label-free density difference amplification-based cell sorting (dDACS) without using any external optical, magnetic, electrical forces, or fluidic activations. The conceptual microfluidic design consists of an inlet, hydraulic jump cavity, and multiple outlets. Incoming particles experience gravity, buoyancy, and drag forces in the separation chamber. The height and distance that each particle can reach in the chamber are different and depend on its density, thus allowing for the separation of particles into multiple outlets. The separation behavior of the particles, based on the ratio of the channel heights of the inlet and chamber and Reynolds number has been systematically studied. Numerical simulation reveals that the difference between the heights of only lighter particles with densities close to that of water increases with increasing the ratio of the channel heights, while decreasing Reynolds number can amplify the difference in the heights between the particles considered irrespective of their densities. PMID:25553185

  15. Immunomicrospheres - Reagents for cell labeling and separation

    NASA Technical Reports Server (NTRS)

    Rembaum, A.; Dreyer, W. J.

    1980-01-01

    Immunomicrospheres are specially designed microscopic particles that have antibodies or similar molecules chemically bound to their surfaces. The antibody-coated microspheres react in a highly specific way with target cells, viruses, or other antigenic agents. Immunomicrospheres may be synthesized so that they incorporate compounds that are highly radioactive, intensely fluorescent, magnetic, electron opaque, highly colored, or pharmacologically active. These various types of microspheres may be coated with pure, highly specific monoclonal antibodies obtained by the new hybridoma cell cloning techniques or with conventional antibody preparations. Some of the many present and potential applications for these new reagents are (1) new types of radioimmune or immunofluorescent assays, (2) improved fluorescence microscopy, (3) separation of cells on the basis of the fluorescent, electrophoretic, or magnetic properties of bound immunomicrospheres, (4) markers for use in several types of electron or standard light microscopy, and (5) delivery of lethal compouds to specific undesirable living cells. The combination of the various new types of synthetic microspheres and the newly available homogeneous antibodies offers new opportunities in research, diagnosis, and therapy.

  16. Technetium-99m-labeled red blood cell imaging

    SciTech Connect

    Front, D.; Israel, O.; Groshar, D.; Weininger, J.

    1984-07-01

    Red blood cells labeled with 99mTc constitute a suitable intravascular agent for imaging of vascular abnormalities. Hemangiomas are characterized by low perfusion and a high blood pool. This ''perfusion blood-pool mismatch,'' not encountered in other lesions, may help in the specific diagnosis of this tumor. This is particularly so in cavernous hemangiomas of the liver where three-phase 99mTc-labeled red blood cell scintigraphy should precede liver biopsy. Red cell scintigraphy also is useful for establishing the vascular nature of hemangiomas of the head and neck and the skin and for diagnosis of venous occlusion. Heat-damaged red blood cells provide a specific spleen imaging agent. This should be used when patients with suspected splenic pathology have equivocal colloid scintigraphy.

  17. Cancer cell labeling and tracking using fluorescent and magnetic nanodiamond.

    PubMed

    Lien, Zhi-Yi; Hsu, Tzu-Chia; Liu, Kuang-Kai; Liao, Wei-Siang; Hwang, Kuo-Chu; Chao, Jui-I

    2012-09-01

    Nanodiamond, a promising carbon nanomaterial, develops for biomedical applications such as cancer cell labeling and detection. Here, we establish the nanodiamond-bearing cancer cell lines using the fluorescent and magnetic nanodiamond (FMND). Treatment with FMND particles did not significantly induce cytotoxicity and growth inhibition in HFL-1 normal lung fibroblasts and A549 lung cancer cells. The fluorescence intensities and particle complexities were increased in a time- and concentration-dependent manner by treatment with FMND particles in lung cancer cells; however, the existence of FMND particles inside the cells did not alter cellular size distribution. The FMND-bearing lung cancer cells could be separated by the fluorescent and magnetic properties of FMNDs using the flow cytometer and magnetic device, respectively. The FMND-bearing cancer cells were identified by the existence of FMNDs using flow cytometer and confocal microscope analysis. More importantly, the cell morphology, viability, growth ability and total protein expression profiles in the FMND-bearing cells were similar to those of the parental cells. The separated FMND-bearing cells with various generations were cryopreservation for further applications. After re-thawing the FMND-bearing cancer cell lines, the cells still retained the cell survival and growth ability. Additionally, a variety of human cancer types including colon (RKO), breast (MCF-7), cervical (HeLa), and bladder (BFTC905) cancer cells could be used the same strategy to prepare the FMND-bearing cancer cells. These results show that the FMND-bearing cancer cell lines, which reserve the parental cell functions, can be applied for specific cancer cell labeling and tracking.

  18. Effect of misoprostol and cimetidine on gastric cell labeling index

    SciTech Connect

    Fich, A.; Arber, N.; Sestieri, M.; Zajicek, G.; Rachmilewitz, D.

    1985-07-01

    The effect of misoprostol and cimetidine on gastric cell turnover was studied. Endoscopic biopsy specimens of fundic and antral mucosa were obtained from duodenal ulcer patients before and after 4 wk of therapy with cimetidine 1.2 g/day or misoprostol 800 micrograms/day. Biopsy specimens were incubated with (/sup 3/H)thymidine. Glandular column length and number of labeled cells were determined after autoradiography. There was no significant difference in column length of antral or fundic glands before or after therapy with cimetidine and misoprostol. The number of antral and fundic labeled cells was significantly decreased after misoprostol treatment (3.6 +/- 0.3 and 4.6 +/- 0.4, mean +/- SE), as opposed to their respective number before therapy (6.9 +/- 0.5 and 8.3 +/- 0.8) (p less than 0.01). On the other hand, after treatment with cimetidine, the number of antral and fundic labeled cells was significantly higher (11.8 +/- 0.9 and 7.5 +/- 1.0, respectively) as compared with their number before therapy (5.7 +/- 0.5 and 5.6 +/- 0.6, respectively). The decreased gastric cell turnover induced by misoprostol indicates that the trophic effect of prostanoids on gastric mucosa is not due to an increase in cellular kinetics. The increased gastric cell turnover induced by cimetidine may contribute to its therapeutic effect in peptic ulcer disease.

  19. Retrograde labelling of serotonergic projections onto the neuroendocrine bag cells of Aplysia.

    PubMed

    McPherson, D R; Blankenship, J E

    1991-02-25

    Injection of rhodamine-conjugated latex microspheres into the right bag cell cluster of Aplysia brasiliana yielded retrograde labelling of a small number of cells in the cerebral and abdominal ganglia. Subsequent staining for serotonin immunoreactivity demonstrated consistent double-labelling in specific cerebral and abdominal ganglion serotonergic cells. The double-labelled populations were also stained in vivo by prior treatment with 5,7-dihydroxytryptamine. These retrogradely labelled serotonergic neurons may represent sources of inhibitory input to the neuroendocrine bag cells.

  20. Blood cell labelling. Theory and methods: radiation hazards.

    PubMed

    Trott, N G; Akbari, R B

    1984-02-01

    The chief physical properties of the radionuclide In111 are outlined, and compared with those of three other radionuclides, Tc99m, I131 and Cr51 which have similar applications. It is pointed out that the gamma-rays of In111 are appreciably more penetrating in lead than those of Tc99m and the significance of this, both in the use of shielding on syringes and in the effectiveness of lead glass screens is discussed. Examples are given of the dosimetry for In111 labelled cells in humans and it is noted that the absorbed dose in the spleen per mCi (37 MBq) injected may be some 10 rad (0.1 Gy). The problems that have been noted of damage to cells arising from oxine labelling and now considered to be due to radiation damage are briefly reviewed. PMID:6719926

  1. Cell Labeling and Injection in Developing Embryonic Mouse Hearts

    PubMed Central

    Dirschinger, Ralf J.; Evans, Sylvia M.; Puceat, Michel

    2014-01-01

    Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development.  Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development. PMID:24797676

  2. Labeling Cytosolic Targets in Live Cells with Blinking Probes

    PubMed Central

    Xu, Jianmin; Chang, Jason; Yan, Qi; Dertinger, Thomas; Bruchez, Marcel; Weiss, Shimon

    2013-01-01

    With the advent of superresolution imaging methods, fast dynamic imaging of biological processes in live cells remains a challenge. A subset of these methods requires the cellular targets to be labeled with spontaneously blinking probes. The delivery and specific targeting of cytosolic targets and the control of the probes’ blinking properties are reviewed for three types of blinking probes: quantum dots, synthetic dyes, and fluorescent proteins. PMID:23930154

  3. Ultra-fast stem cell labelling using cationised magnetoferritin

    NASA Astrophysics Data System (ADS)

    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

  4. Label-free cell separation and sorting in microfluidic systems

    PubMed Central

    Gossett, Daniel R.; Weaver, Westbrook M.; Mach, Albert J.; Hur, Soojung Claire; Tse, Henry Tat Kwong; Lee, Wonhee; Amini, Hamed

    2010-01-01

    Cell separation and sorting are essential steps in cell biology research and in many diagnostic and therapeutic methods. Recently, there has been interest in methods which avoid the use of biochemical labels; numerous intrinsic biomarkers have been explored to identify cells including size, electrical polarizability, and hydrodynamic properties. This review highlights microfluidic techniques used for label-free discrimination and fractionation of cell populations. Microfluidic systems have been adopted to precisely handle single cells and interface with other tools for biochemical analysis. We analyzed many of these techniques, detailing their mode of separation, while concentrating on recent developments and evaluating their prospects for application. Furthermore, this was done from a perspective where inertial effects are considered important and general performance metrics were proposed which would ease comparison of reported technologies. Lastly, we assess the current state of these technologies and suggest directions which may make them more accessible. Figure A wide range of microfluidic technologies have been developed to separate and sort cells by taking advantage of differences in their intrinsic biophysical properties PMID:20419490

  5. Labeling and imaging cells in the zebrafish hindbrain.

    PubMed

    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. Commercial Nanoparticles for Stem Cell Labeling and Tracking

    PubMed Central

    Wang, Yaqi; Xu, Chenjie; Ow, Hooisweng

    2013-01-01

    Stem cell therapy provides promising solutions for diseases and injuries that conventional medicines and therapies cannot effectively treat. To achieve its full therapeutic potentials, the homing process, survival, differentiation, and engraftment of stem cells post transplantation must be clearly understood. To address this need, non-invasive imaging technologies based on nanoparticles (NPs) have been developed to track transplanted stem cells. Here we summarize existing commercial NPs which can act as contrast agents of three commonly used imaging modalities, including fluorescence imaging, magnetic resonance imaging and photoacoustic imaging, for stem cell labeling and tracking. Specifically, we go through their technologies, industry distributors, applications and existing concerns in stem cell research. Finally, we provide an industry perspective on the potential challenges and future for the development of new NP products. PMID:23946821

  7. Label-free electronic detection of target cells

    NASA Astrophysics Data System (ADS)

    Esfandyarpour, Rahim; Javanmard, Mehdi; Harris, James; Davis, Ronald W.

    2014-03-01

    In this manuscript we describe an electronic label-free method for detection of target cells, which has potential applications ranging from pathogen detection for food safety all the way to detection of circulating tumor cells for cancer diagnosis. The nanoelectronic platform consists of a stack of electrodes separated by a 30nm thick insulating layer. Cells binding to the tip of the sensor result in a decrease in the impedance at the sensing tip due to an increase in the fringing capacitance between the electrodes. As a proof of concept we demonstrate the ability to detect Saccharomyces Cerevisae cells with high specificity using a sensor functionalized with Concanavalin A. Ultimately we envision using this sensor in conjunction with a technology for pre-concentration of target cells to develop a fully integrated micro total analysis system.

  8. Functionalized nanopipettes: toward label-free, single cell biosensors

    PubMed Central

    Actis, Paolo; Mak, Andy C.

    2010-01-01

    Nanopipette technology has been proven to be a label-free biosensor capable of identifying DNA and proteins. The nanopipette can include specific recognition elements for analyte discrimination based on size, shape, and charge density. The fully electrical read-out and the ease and low-cost fabrication are unique features that give this technology an enormous potential. Unlike other biosensing platforms, nanopipettes can be precisely manipulated with submicron accuracy and used to study single cell dynamics. This review is focused on creative applications of nanopipette technology for biosensing. We highlight the potential of this technology with a particular attention to integration of this biosensor with single cell manipulation platforms. PMID:20730113

  9. Labeling index in squamous cell carcinoma of the larynx

    SciTech Connect

    Balzi, M.; Ninu, B.M.; Becciolini, A.; Scubla, E.; Boanini, P.; Gallina, E.; Gallo, O.; Fini-Storchi, O.; Bondi, R. )

    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.

  10. Hoechst fluorescence intensity can be used to separate viable bromodeoxyuridine-labeled cells from viable non-bromodeoxyuridine-labeled cells

    NASA Technical Reports Server (NTRS)

    Mozdziak, P. E.; Pulvermacher, P. M.; Schultz, E.; Schell, K.

    2000-01-01

    BACKGROUND: 5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a cell. Most techniques that identify BrdU-labeled cells require conditions that kill the cells. However, the fluorescence intensity of the membrane-permeable Hoechst dyes is reduced by the incorporation of BrdU into DNA, allowing the separation of viable BrdU positive (BrdU+) cells from viable BrdU negative (BrdU-) cells. METHODS: Cultures of proliferating cells were supplemented with BrdU for 48 h and other cultures of proliferating cells were maintained without BrdU. Mixtures of viable BrdU+ and viable BrdU- cells from the two proliferating cultures were stained with Hoechst 33342. The viable BrdU+ and BrdU- cells were sorted into different fractions from a mixture of BrdU+ and BrdU- cells based on Hoechst fluorescence intensity and the ability to exclude the vital dye, propidium iodide. Subsequently, samples from the original mixture, the sorted BrdU+ cell population, and the sorted BrdU- cell population were immunostained using an anti-BrdU monoclonal antibody and evaluated using flow cytometry. RESULTS: Two mixtures consisting of approximately 55% and 69% BrdU+ cells were sorted into fractions consisting of greater than 93% BrdU+ cells and 92% BrdU- cells. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. CONCLUSIONS: Hoechst fluorescence intensity in combination with cell sorting is an effective tool to separate viable BrdU+ from viable BrdU- cells for further study. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. Copyright 2000 Wiley-Liss, Inc.

  11. Functionalized magnetic-fluorescent hybrid nanoparticles for cell labelling.

    PubMed

    Lou, Lei; Yu, Ke; Zhang, Zhengli; Li, Bo; Zhu, Jianzhong; Wang, Yiting; Huang, Rong; Zhu, Ziqiang

    2011-05-01

    A facile method of synthesizing 60 nm magnetic-fluorescent core-shell bifunctional nanocomposites with the ability to label cells is presented. Hydrophobic trioctylphosphine oxide (TOPO)-capped CdSe@ZnS quantum dots (QDs) were assembled on polyethyleneimine (PEI)-coated Fe(3)O(4) nanoparticles (MNP). Polyethyleneimine was utilized for the realization of multifunction, including attaching 4 nm TOPO capped CdSe@ZnS quantum dots onto magnetite particles, altering the surface properties of quantum dots from hydrophobic to hydrophilic as well as preventing the formation of large aggregates. Results show that these water-soluble hybrid nanocomposites exhibit good colloidal stability and retain good magnetic and fluorescent properties. Because TOPO-capped QDs are assembled instead of their water-soluble equivalents, the nanocomposites are still highly luminescent with no shift in the PL peak position and present long-term fluorescence stability. Moreover, TAT peptide (GRKKRRQRRRPQ) functionalized hybrid nanoparticles were also studied due to their combined magnetic enrichment and optical detection for cell separation and rapid cell labelling. A cell viability assay revealed good biocompatibility of these hybrid nanoparticles. The potential application of the new magnetic-fluorescent nanocomposites in biological and medicine is demonstrated. PMID:21503355

  12. Computational cell analysis for label-free detection of cell properties in a microfluidic laminar flow.

    PubMed

    Zhang, Alex Ce; Gu, Yi; Han, Yuanyuan; Mei, Zhe; Chiu, Yu-Jui; Geng, Lina; Cho, Sung Hwan; Lo, Yu-Hwa

    2016-06-20

    Although a flow cytometer, being one of the most popular research and clinical tools for biomedicine, can analyze cells based on the cell size, internal structures such as granularity, and molecular markers, it provides little information about the physical properties of cells such as cell stiffness and physical interactions between the cell membrane and fluid. In this paper, we propose a computational cell analysis technique using cells' different equilibrium positions in a laminar flow. This method utilizes a spatial coding technique to acquire the spatial position of the cell in a microfluidic channel and then uses mathematical algorithms to calculate the ratio of cell mixtures. Most uniquely, the invented computational cell analysis technique can unequivocally detect the subpopulation of each cell type without labeling even when the cell type shows a substantial overlap in the distribution plot with other cell types, a scenario limiting the use of conventional flow cytometers and machine learning techniques. To prove this concept, we have applied the computation method to distinguish live and fixed cancer cells without labeling, count neutrophils from human blood, and distinguish drug treated cells from untreated cells. Our work paves the way for using computation algorithms and fluidic dynamic properties for cell classification, a label-free method that can potentially classify over 200 types of human cells. Being a highly cost-effective cell analysis method complementary to flow cytometers, our method can offer orthogonal tests in companion with flow cytometers to provide crucial information for biomedical samples. PMID:27163941

  13. Dependence of technetium-99m red blood cell labeling efficiency on red cell surface charge.

    PubMed

    Seldin, D W; Simchon, S; Jan, K M; Chien, S; Alderson, P O

    1988-10-01

    The mechanisms by which [99mTc]pertechnetate becomes attached to stannous-primed red blood cells are not known in detail. To study the problem further, the effect of red cell surface charge on labeling efficiency was evaluated. Red cell surface charge was reduced by using the enzyme neuraminidase to remove the terminal charge-bearing sialic acid moiety of the membrane glycoprotein. Forty-five blood samples from six volunteers were treated with neuraminidase for varying lengths of time, resulting in the removal of from 11% to 99% of the normal negative surface charge, as determined from electrophoretic mobility measurements. There was excellent linear correlation between labeling efficiency and the remaining red cell surface charge for values down to 20% of normal (r = 0.89). When surface charge was less than 20% of normal, labeling efficiency was constant at 30%. Eleven blood samples from three donors were divided into two groups that were treated with neuraminidase either before or after they were labeled. The labeling efficiency was independent of the order in which the steps were performed. No evidence for shifting of the radiolabel from the cell membrane to hemoglobin was found. The results suggest that clinical conditions associated with a reduction of sialic acid on the erythrocyte membrane may be one cause of decreased red blood cell labeling efficiency, and that increased membrane permeability for reduced technetium species may be responsible for the decrease.

  14. Live-cell protein labelling with nanometre precision by cell squeezing

    PubMed Central

    Kollmannsperger, Alina; Sharei, Armon; Raulf, Anika; Heilemann, Mike; Langer, Robert; Jensen, Klavs F.; Wieneke, Ralph; Tampé, Robert

    2016-01-01

    Live-cell labelling techniques to visualize proteins with minimal disturbance are important; however, the currently available methods are limited in their labelling efficiency, specificity and cell permeability. We describe high-throughput protein labelling facilitated by minimalistic probes delivered to mammalian cells by microfluidic cell squeezing. High-affinity and target-specific tracing of proteins in various subcellular compartments is demonstrated, culminating in photoinduced labelling within live cells. Both the fine-tuned delivery of subnanomolar concentrations and the minimal size of the probe allow for live-cell super-resolution imaging with very low background and nanometre precision. This method is fast in probe delivery (∼1,000,000 cells per second), versatile across cell types and can be readily transferred to a multitude of proteins. Moreover, the technique succeeds in combination with well-established methods to gain multiplexed labelling and has demonstrated potential to precisely trace target proteins, in live mammalian cells, by super-resolution microscopy. PMID:26822409

  15. Nanoscale Label-free Bioprobes to Detect Intracellular Proteins in Single Living Cells

    PubMed Central

    Hong, Wooyoung; Liang, Feng; Schaak, Diane; Loncar, Marko; Quan, Qimin

    2014-01-01

    Fluorescent labeling techniques have been widely used in live cell studies; however, the labeling processes can be laborious and challenging for use in non-transfectable cells, and labels can interfere with protein functions. While label-free biosensors have been realized by nanofabrication, a method to track intracellular protein dynamics in real-time, in situ and in living cells has not been found. Here we present the first demonstration of label-free detection of intracellular p53 protein dynamics through a nanoscale surface plasmon-polariton fiber-tip-probe (FTP). PMID:25154394

  16. Nanoscale Label-free Bioprobes to Detect Intracellular Proteins in Single Living Cells

    NASA Astrophysics Data System (ADS)

    Hong, Wooyoung; Liang, Feng; Schaak, Diane; Loncar, Marko; Quan, Qimin

    2014-08-01

    Fluorescent labeling techniques have been widely used in live cell studies; however, the labeling processes can be laborious and challenging for use in non-transfectable cells, and labels can interfere with protein functions. While label-free biosensors have been realized by nanofabrication, a method to track intracellular protein dynamics in real-time, in situ and in living cells has not been found. Here we present the first demonstration of label-free detection of intracellular p53 protein dynamics through a nanoscale surface plasmon-polariton fiber-tip-probe (FTP).

  17. Quantum dot-labeled aptamer nanoprobes specifically targeting glioma cells

    NASA Astrophysics Data System (ADS)

    Chen, Xue-Chai; Deng, Yu-Lin; Lin, Yi; Pang, Dai-Wen; Qing, Hong; Qu, Feng; Xie, Hai-Yan

    2008-06-01

    Two new techniques, aptamer-based specific recognition and quantum dot (QD)-based fluorescence labeling, are becoming increasingly important in biosensing. In this study, these two techniques have been coupled together to construct a new kind of fluorescent QD-labeled aptamer (QD-Apt) nanoprobe by conjugating GBI-10 aptamer to the QD surface. GBI-10 is a single-stranded DNA (ssDNA) aptamer for tenascin-C, which distributes on the surface of glioma cells as a dominant extracellular matrix protein. The QD-Apt nanoprobe can recognize the tenascin-C on the human glioma cell surface, which will be helpful for the development of new convenient and sensitive in vitro diagnostic assays for glioma. The QD-Apt nanoprobe has particular features such as strong fluorescence, stability, monodispersity and uniformity. In addition, this probe preparation method is universal, so it is expected to provide a new type of stable nanoprobe for high-throughput and fast biosensing detection and bioimaging. New methods for real-time and dynamic tracking and imaging can be accordingly developed.

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

    PubMed Central

    El-Sadik, Abir O; El-Ansary, Afaf; Sabry, Sherif M

    2010-01-01

    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. PMID:22291483

  19. Cell-selective labelling of proteomes in Drosophila melanogaster

    PubMed Central

    Erdmann, Ines; Marter, Kathrin; Kobler, Oliver; Niehues, Sven; Abele, Julia; Müller, Anke; Bussmann, Julia; Storkebaum, Erik; Ziv, Tamar; Thomas, Ulrich; Dieterich, Daniela C.

    2015-01-01

    The specification and adaptability of cells rely on changes in protein composition. Nonetheless, uncovering proteome dynamics with cell-type-specific resolution remains challenging. Here we introduce a strategy for cell-specific analysis of newly synthesized proteomes by combining targeted expression of a mutated methionyl-tRNA synthetase (MetRS) with bioorthogonal or fluorescent non-canonical amino-acid-tagging techniques (BONCAT or FUNCAT). Substituting leucine by glycine within the MetRS-binding pocket (MetRSLtoG) enables incorporation of the non-canonical amino acid azidonorleucine (ANL) instead of methionine during translation. Newly synthesized proteins can thus be labelled by coupling the azide group of ANL to alkyne-bearing tags through ‘click chemistry'. To test these methods for applicability in vivo, we expressed MetRSLtoG cell specifically in Drosophila. FUNCAT and BONCAT reveal ANL incorporation into proteins selectively in cells expressing the mutated enzyme. Cell-type-specific FUNCAT and BONCAT, thus, constitute eligible techniques to study protein synthesis-dependent processes in complex and behaving organisms. PMID:26138272

  20. Correlative fluorescence and electron microscopy of quantum dot labeled proteins on whole cells in liquid.

    PubMed

    Peckys, Diana B; Dukes, Madeline J; de Jonge, Niels

    2014-01-01

    Correlative fluorescence microscopy and scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot (QD) nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, the microchip with the labeled cells and one with a spacer are assembled in a special microfluidic device and imaged with STEM.

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

    NASA Astrophysics Data System (ADS)

    Liu, Jianbo; Zhang, Pengfei; Yang, Xiaohai; Wang, Kemin; Guo, Qiuping; Huang, Jin; Li, Wei

    2014-12-01

    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.

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

    PubMed

    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.

  3. Superparamagnetic iron oxide nanoparticles for direct labeling of stem cells and in vivo MRI tracking.

    PubMed

    Kim, Saejeong J; Lewis, Bobbi; Steiner, Mark-Steven; Bissa, Ursula V; Dose, Christian; Frank, Joseph A

    2016-01-01

    To develop effective stem cell therapies, it is important to track therapeutic cells non-invasively and monitor homing to areas of pathology. The purpose of this study was to design and evaluate the labeling efficiency of commercially available dextran-coated superparamagnetic iron oxide nanoparticles, FeraTrack Direct (FTD), in various stem and immune cells; assess the cytotoxicity and tolerability of the FTD in stem cells; and monitor stem cell homing using FTD-labeled bone-marrow-derived mesenchymal stromal cells (BMSCs) and neural stem cells (NSCs) in a tumor model by in vivo MRI. BMSCs, NSCs, hematopoietic stem cells (HSCs), T-lymphocytes, and monocytes were labeled effectively with FTD without the need for transfection agents, and Prussian blue (PB) staining and transmission electron microscopy (TEM) confirmed intracellular uptake of the agent. The viability, proliferation, and functionality of the labeled cells were minimally or not affected after labeling. When 10(6) FTD-labeled BMSCs or NSCs were injected into C6 glioma bearing nude mice, the cells homing to the tumors were detected as hypointense regions within the tumor using 3 T clinical MRI up to 10 days post injection. Histological analysis confirmed the homing of injected cells to the tumor by the presence of PB positive cells that are not macrophages. Labeling of stem cells or immune cells with FTD was non-toxic, and should facilitate the translation of this agent to clinical trials for evaluation of trafficking of cells by MRI.

  4. Long-term effect of vital labelling on mixed Schwann cell cultures.

    PubMed

    Mosahebi, A; Woodward, B; Green, C; Martin, R; Terenghi, G

    2000-06-01

    Schwann cell transplantation following neuronal injury could encourage regeneration of spinal cord as well as improving peripheral nerve gap repair. In order to gain a better understanding of the role of transplanted Schwann cells in vivo, it is essential to be able to follow their behaviour after transplantation. Our aim was to evaluate the suitability of two vital fluorescent labels on the proliferation rate and phenotypic stability of Schwann cells, in either pure culture or mixed co-culture. Primary cultures of Schwann cells were obtained from Dark Agouti and Lewis neonatal rats and labelled with H33342 and PKH26, respectively. In mixed cultures, a 50: 50 mixture of Dark Agouti and Lewis Schwann cells was present. Labelled cultured cells were examined at 1, 2 and 4 weeks for viability and phenotypic marker expression of S100, GFAP, p75, MHC I, MHC II and compared with corresponding unlabelled cells. The results showed that although there was no deleterious interaction in the mixed cultures, the viability was reduced by the labelling after 2 weeks. Labelled cells could be distinguished up to 4 weeks, but there was leakage of H33342 label after 2 weeks. Labelled Schwann cells showed reduced expression of phenotypic markers, especially p75 when labelled with H33342. In conclusion, H33342 and PKH26 can be used as fluorescent markers of Schwann cells for short-term studies, for a maximum of 2 weeks, but different markers may be needed for longer experiments.

  5. Radionuclide-labeled red blood cells: current status and future prospects

    SciTech Connect

    Srivastava, S.C.; Chervu, L.R.

    1984-04-01

    Radiolabeling of red cells and their clinical and research application in nuclear medicine constitute an area of continued interest and steady growth during the past two decades. Technetium-/sup 99/m-labeled red cells in particular have revolutionized the field of cardiovascular nuclear medicine by making possible the external evaluation of various heart parameters with minimum radiation dose or trauma to the patient. Among other areas of study that use /sup 99/mTc -RBC are blood pool imaging, detection of vascular malformations, red cell mass determination, detection of gastrointestinal bleeding, and of hemangiomas. Heat-damaged /sup 99/mTc -RBC find application in spleen imaging, accessory spleen localization, detection of GI bleeding, and in other areas. A critical evaluation is presented of the various in vitro and in vivo labeling techniques that are currently available for red cell labeling. Even though the presently used procedures provide satisfactory labeled preparations, ideal radioisotopic RBC labels remain to be developed. Intermediate (2-3 days) as well as long-lived (approximately 30 days) radionuclidic labels are highly desirable for a number of clinical procedures where /sup 99/mTc is not useful due to its short half-life. New approaches such as the use of radiolabeled antibodies to red cell antigens, or labeling specific receptor sites in the cell may lead to substantial improvements in the labeling methodology and could yield labeled cells with the least damage and maximum in vivo stability.

  6. Enhanced detection of fluorescence quenching in labeled cells

    DOEpatents

    Crissman, Harry A.; Steinkamp, John A.

    1992-01-01

    A method is provided for quantifying BrdU labeled DNA in cells. The BrdU is incorporated into the DNA and the DNA is stained with a first fluorochrome having a fluorescence which is quenchable by BrdU. The first fluorochrome is preferably a thymidine base halogen analogue, such as a Hoechst fluorochrome. The DNA is then stained with a second fluorochrome having a fluorescence that is substantially uneffected by BrdU. The second fluorochrome may be selected from the group consisting of mithramycin, chromomycin A3, olivomycin, propidium iodide and ethidium bromine. The fluorescence from the first and second fluorochromes is then measured to obtain first and second output signals, respectively. The first output signal is substracted from the second output signal to obtain a difference signal which is functionally related to the quantity of BrdU incorporated into DNA. The technique is particularly useful for quantifying the synthesis of DNA during the S-phase of the cell cycle.

  7. Enhanced detection of fluorescence quenching in labeled cells

    DOEpatents

    Crissman, H.A.; Steinkamp, J.A.

    1987-11-30

    A method is provided for quantifying BrdU labeled DNA in cells. The BrdU is substituted onto the DNA and the DNA is stained with a first fluorochrome having a fluorescence which is quenchable by BrdU. The first fluorochrome is preferably a thymidine base halogen analogue, such as a Hoechst fluorochrome. The DNA is then stained with a second fluorochrome having a fluorescence which is substantially uneffected by BrdU. The second fluorochrome may be selected from the group consisting of mithramycin, chromomycin A3, olivomycin, propidium iodide and ethidium bromine. The fluorescence from the first and second fluorochromes is then measured to obtain first and second output signals, respectively. The first output signal is subtracted from the second output signal to obtain a difference signal which is functionally related to the quantity of BrdU incorporated into DNA. The technique is particularly useful for quantifying the synthesis of DNA during the S-phase of the cell cycle. 2 figs.

  8. Identification of Putative Bovine Mammary Epithelial Stem Cells by Their Retention of Labeled DNA Strands

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Stem cells characteristically retain labeled DNA for extended periods due to their selective segregation of template DNA strands during mitosis. In this study, proliferating cells in the prepubertal bovine mammary gland were labeled using five daily-injections of 5-bromo-2-deoxyuridine (BrdU). Fiv...

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

    PubMed

    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

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

    NASA Astrophysics Data System (ADS)

    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.

  11. Retrograde labeling, enrichment, and characterization of retinal ganglion cells from the neonatal rat.

    PubMed

    Sarthy, P V; Curtis, B M; Catterall, W A

    1983-12-01

    We have developed a method for labeling retinal ganglion cells in neonatal rats by retrograde transport of the fluorescent dye, True Blue (TB), injected into the optic chiasm. Following proteolytic dissociation of labeled retinas into single cells, the labeled cells could be enriched 50- to 100-fold by centrifugation in a 5%/10% metrizamide gradient. When plated in Ham's F-10 medium in the presence of fetal calf serum and chick optic tectum-conditioned medium, the labeled cells could be maintained in vitro up to 48 hr. In these cultures, the ganglion cells (GCS) constituted 50 to 70% of the total cell population. When GC-rich fractions or GC cultures were stained with a monoclonal antibody to Thy-1 antigen, greater than 90% of the TB-labeled cells were reactive. In order to localize voltage-sensitive sodium channels, GC-rich cultures were reacted with 125I-scorpion toxin. Analysis of the autoradiograms showed that the density of silver grains was about 10-fold higher on TB-labeled cells than on nonfluorescent cells, or in controls which contained excess of unlabeled toxin. When GC cultures were incubated with micromolar concentrations of putative GC transmitters, aspartate and glutamate, the amino acids were accumulated by 15 to 20% of labeled cells. Several lectin receptors were also localized on TB-labeled cells in situ. Whereas the lectins wheat germ agglutinin, concanavalin A, peanut agglutinin, Dolichos biflorus agglutinin, and Limulus polyphemus agglutinin bound to TB-labeled cells, others such as Ricinus communis agglutinin I, Ulex, and Lotus lectins showed no binding. The lectin binding was specific since preincubation with the appropriate hapten sugar blocked lectin binding.

  12. Cell Labeling and Tracking Method without Distorted Signals by Phagocytosis of Macrophages

    PubMed Central

    Kang, Sun-Woong; Lee, Sangmin; Na, Jin Hee; Yoon, Hwa In; Lee, Dong-Eun; Koo, Heebeom; Cho, Yong Woo; Kim, Sun Hwa; Jeong, Seo Young; Kwon, Ick Chan; Choi, Kuiwon; Kim, Kwangmeyung

    2014-01-01

    Cell labeling and tracking are important processes in understanding biologic mechanisms and the therapeutic effect of inoculated cells in vivo. Numerous attempts have been made to label and track inoculated cells in vivo; however, these methods have limitations as a result of their biological effects, including secondary phagocytosis of macrophages and genetic modification. Here, we investigated a new cell labeling and tracking strategy based on metabolic glycoengineering and bioorthogonal click chemistry. We first treated cells with tetra-acetylated N-azidoacetyl-D-mannosamine to generate unnatural sialic acids with azide groups on the surface of the target cells. The azide-labeled cells were then transplanted to mouse liver, and dibenzyl cyclooctyne-conjugated Cy5 (DBCO-Cy5) was intravenously injected into mice to chemically bind with the azide groups on the surface of the target cells in vivo for target cell visualization. Unnatural sialic acids with azide groups could be artificially induced on the surface of target cells by glycoengineering. We then tracked the azide groups on the surface of the cells by DBCO-Cy5 in vivo using bioorthogonal click chemistry. Importantly, labeling efficacy was enhanced and false signals by phagocytosis of macrophages were reduced. This strategy will be highly useful for cell labeling and tracking. PMID:24578725

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

    PubMed Central

    Ariza de Schellenberger, Angela; Kratz, Harald; Farr, Tracy D; Löwa, Norbert; Hauptmann, Ralf; Wagner, Susanne; Taupitz, Matthias; Schnorr, Jörg; Schellenberger, Eyk A

    2016-01-01

    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-uptake-dependent biocompatibility studies and cell detection by MRI and future MPI. Additionally, using a 7 T MR imager equipped with a cryocoil resulted in approximately two times higher detection. In conclusion, we established labeling conditions for new high-relaxivity MCP, VSOP, and Resovist® for improved MRI of MSC with single-cell sensitivity. PMID:27110112

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

    PubMed

    Ariza de Schellenberger, Angela; Kratz, Harald; Farr, Tracy D; Löwa, Norbert; Hauptmann, Ralf; Wagner, Susanne; Taupitz, Matthias; Schnorr, Jörg; Schellenberger, Eyk A

    2016-01-01

    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-uptake-dependent biocompatibility studies and cell detection by MRI and future MPI. Additionally, using a 7 T MR imager equipped with a cryocoil resulted in approximately two times higher detection. In conclusion, we established labeling conditions for new high-relaxivity MCP, VSOP, and Resovist(®) for improved MRI of MSC with single-cell sensitivity. PMID:27110112

  15. New Red-Emitting Tetrazine-Phenoxazine Fluorogenic Labels for Live-Cell Intracellular Bioorthogonal Labeling Schemes.

    PubMed

    Knorr, Gergely; Kozma, Eszter; Herner, András; Lemke, Edward A; Kele, Péter

    2016-06-20

    The synthesis of a set of tetrazine-bearing fluorogenic dyes suitable for intracellular labeling of proteins in live cells is presented. The red excitability and emission properties ensure minimal autofluorescence, while through-bond energy-transfer-based fluorogenicity reduces nonspecific background fluorescence of unreacted dyes. The tetrazine motif efficiently quenches fluorescence of the phenoxazine core, which can be selectively turned on chemically upon bioorthogonal inverse-electron-demand Diels-Alder reaction with proteins modified genetically with strained trans-cyclooctenes.

  16. Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

    PubMed Central

    Rickert, Christof; Kunz, Thomas; Harris, Kerri-Lee; Whitington, Paul; Technau, Gerhard

    2013-01-01

    In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution. PMID:23486245

  17. Classification of blood cells and tumor cells using label-free ultrasound and photoacoustics.

    PubMed

    Strohm, Eric M; Kolios, Michael C

    2015-08-01

    A label-free method that can identify cells in a blood sample using high frequency photoacoustic and ultrasound signals is demonstrated. When the wavelength of the ultrasound or photoacoustic wave is similar to the size of a single cell (frequencies of 100-500 MHz), unique periodic features occur within the ultrasound and photoacoustic power spectrum that depend on the cell size, structure, and morphology. These spectral features can be used to identify different cell types present in blood, such as red blood cells (RBCs), white blood cells (WBCs), and circulating tumor cells. Circulating melanoma cells are ideal for photoacoustic detection due to their endogenous optical absorption properties. Using a 532 nm pulsed laser and a 375 MHz transducer, the ultrasound and photoacoustic signals from RBCs, WBCs, and melanoma cells were individually measured in an acoustic microscope to examine how the signals change between cell types. A photoacoustic and ultrasound signal was detected from RBCs and melanoma cells; only an ultrasound signal was detected from WBCs. The different cell types were distinctly separated using the ultrasound and photoacoustic signal amplitude and power spectral periodicity. The size of each cell was also estimated from the spectral periodicity. For the first time, sound waves generated using pulse-echo ultrasound and photoacoustics have been used to identify and size single cells, with applications toward counting and identifying cells, including circulating melanoma cells. PMID:26079610

  18. Classification of blood cells and tumor cells using label-free ultrasound and photoacoustics.

    PubMed

    Strohm, Eric M; Kolios, Michael C

    2015-08-01

    A label-free method that can identify cells in a blood sample using high frequency photoacoustic and ultrasound signals is demonstrated. When the wavelength of the ultrasound or photoacoustic wave is similar to the size of a single cell (frequencies of 100-500 MHz), unique periodic features occur within the ultrasound and photoacoustic power spectrum that depend on the cell size, structure, and morphology. These spectral features can be used to identify different cell types present in blood, such as red blood cells (RBCs), white blood cells (WBCs), and circulating tumor cells. Circulating melanoma cells are ideal for photoacoustic detection due to their endogenous optical absorption properties. Using a 532 nm pulsed laser and a 375 MHz transducer, the ultrasound and photoacoustic signals from RBCs, WBCs, and melanoma cells were individually measured in an acoustic microscope to examine how the signals change between cell types. A photoacoustic and ultrasound signal was detected from RBCs and melanoma cells; only an ultrasound signal was detected from WBCs. The different cell types were distinctly separated using the ultrasound and photoacoustic signal amplitude and power spectral periodicity. The size of each cell was also estimated from the spectral periodicity. For the first time, sound waves generated using pulse-echo ultrasound and photoacoustics have been used to identify and size single cells, with applications toward counting and identifying cells, including circulating melanoma cells.

  19. Synthesis of a fluorine-18 labeled hypoxic cell sensitizer

    SciTech Connect

    Jerabek, P.A.; Dischino, D.D.; Kilbourn, M.R.; Welch, M.J.

    1984-01-01

    The objective of this work was to synthesize a positron emitting radiosensitizing agent as a potential in vivo marker of hypoxic regions within tumors, and ischemic areas of the heart and brain. The method involved radiochemical synthesis of fluorine-18 labeled 1-(2-nitro-imidazolyl)-3-fluoro-2-propanol via nucleophilic ring opening of 1-(2,3-epoxypropyl)2-nitro-imidzole by fluorine-18 labeled tetrabutylammonium fluoride (TBAF). Fluroine-18 TBAF was prepared by the exchange reaction of TBAF with aqueous flourine-18 produced by proton bombardment of enriched oxygen-18 water. The aqueous solution was evaporated carefully by azeotropic distillation with acetonitrile. The fluorine-18 labeled TBAF was taken up in N,N-dimethylacetamide or dimethysulfoxide, then reacted with the episode at 60C for 30 minutes. Separation and identification of the fluorine-18 labeled products by high performance liquid chromatography showed a radioactive peak with a retention time identical to that of 1-(2-nitro-1-imidazolyl)-3-fluoro-2-propanol and a second radioactive peak with a retention time three minutes longer in addition to unreacted fluorine-18 labeled TBAF. The second radioactive peak may represent fluorine-18 labeled 1-2-nitro-1-imidazolyl)-2-fluoro-3-propanol. The average radiochemical yield from reactions run in N,N-dimethylacetamide using 20 micromoles of TBAF and 1-2 mg of the epoxide was l7% in a synthesis time of about 40 minutes. The synthesis of fluorohydrins by the reaction of fluorine-18 labeled TBAF on epoxides represents a new method for the preparation of fluorine-18 labeled fluorohydrins.

  20. Gallbladder visualization during technetium-99m-labeled red cell scintigraphy for gastrointestinal bleeding

    SciTech Connect

    Brill, D.R.

    1985-12-01

    Localization of radionuclide activity in the gallbladder was seen on delayed views following injection of 99mTc-labeled red blood cells for gastrointestinal bleeding in five patients. The mechanism for this unusual finding probably relates to labeling of heme, the biochemical precursor of bilirubin. All patients had had prior transfusions. All but one had severe renal impairment, probably an important predisposing factor.

  1. GABAergic and glycinergic pathways to goldfish retinal ganglion cells: an ultrastructural double label study

    SciTech Connect

    Muller, J.F.

    1987-01-01

    An ultrastructural double label has been employed to compare GABAergic and glycinergic systems in the inner plexiform layer (IPL) of the goldfish retina. Electron microscope autoradiography of /sup 3/H-GABA and /sup 3/H-glycine uptake was combined with retrograde HRP-labeling of ganglion cells. When surveyed for distribution, GABAergic and glycinergic synapses were found onto labeled ganglion cells throughout the IPL. This reinforces previous physiological work that described GABAergic and glycinergic influences on a variety of ganglion cells in goldfish and carp; These physiological effects often reflect direct inputs.

  2. Differentiation of normal and leukemic cells by 2D light scattering label-free static cytometry.

    PubMed

    Xie, Linyan; Liu, Qiao; Shao, Changshun; Su, Xuantao

    2016-09-19

    Two-dimensional (2D) light scattering patterns of single microspheres, normal granulocytes and leukemic cells are obtained by label-free static cytometry. Statistical results of experimental 2D light scattering patterns obtained from standard microspheres with a mean diameter of 4.19 μm agree well with theoretical simulations. High accuracy rates (greater than 92%) for label-free differentiation of normal granulocytes and leukemic cells, both the acute and chronic leukemic cells, are achieved by analyzing the 2D light scattering patterns. Our label-free static cytometry is promising for leukemia screening in clinics. PMID:27661908

  3. Surface-Enhanced Raman Scattering Nanoparticles as Optical Labels for Imaging Cell Surface Proteins

    NASA Astrophysics Data System (ADS)

    MacLaughlin, Christina M.

    Assaying the expression of cell surface proteins has widespread application for characterizing cell type, developmental stage, and monitoring disease transformation. Immunophenotyping is conducted by treating cells with labelled targeting moieties that have high affinity for relevant surface protein(s). The sensitivity and specificity of immunophenotyping is defined by the choice of contrast agent and therefore, the number of resolvable signals that can be used to simultaneously label cells. Narrow band width surface-enhanced Raman scattering (SERS) nanoparticles are proposed as optical labels for multiplexed immunophenotying. Two types of surface coatings were investigated to passivate the gold nanoparticles, incorporate SERS functionality, and to facilitate attachment of targeting antibodies. Thiolated poly(ethylene glycol) forms dative bonds with the gold surface and is compatible with multiple physisorbed Raman-active reporter molecules. Ternary lipid bilayers are used to encapsulate the gold nanoparticles particles, and incorporate three different classes of Raman reporters. TEM, UV-Visible absorbance spectroscopy, DLS, and electrophoretic light scattering were used characterize the particle coating. Colourimetric protein assay, and secondary antibody labelling were used to quantify the antibody conjugation. Three different in vitromodels were used to investigate the binding efficacy and specificity of SERS labels for their biomarker targets. Primary human CLL cells, LY10 B lymphoma, and A549 adenocarcinoma lines were targeted. Dark field imaging was used to visualize the colocalization of SERS labels with cells, and evidence of receptor clustering was obtained based on colour shifts of the particles' Rayleigh scattering. Widefield, and spatially-resolved Raman spectra were used to detect labels singly, and in combination from labelled cells. Fluorescence flow cytometry was used to test the particles' binding specificity, and SERS from labelled cells was also

  4. Labeling pluripotent stem cell-derived neural progenitors with iron oxide particles for magnetic resonance imaging.

    PubMed

    Sart, Sébastien; Bejarano, Fabian Calixto; Yan, Yuanwei; Grant, Samuel C; Li, Yan

    2015-01-01

    Due to the unlimited proliferation capacity and the unique differentiation ability of pluripotent stem cells (PSCs), including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), large numbers of PSC-derived cell products are in demand for applications in drug screening, disease modeling, and especially cell therapy. In stem cell-based therapy, tracking transplanted cells with magnetic resonance imaging (MRI) has emerged as a powerful technique to reveal cell survival and distribution. This chapter illustrated the basic steps of labeling PSC-derived neural progenitors (NPs) with micron-sized particles of iron oxide (MPIO, 0.86 μm) for MRI analysis. The protocol described PSC expansion and differentiation into NPs, and the labeling of the derived cells either after replating on adherent surface or in suspension. The labeled cells can be analyzed using in vitro MRI analysis. The methods presented here can be easily adapted for cell labeling in cell processing facilities under current Good Manufacturing Practices (cGMP). The iron oxide-labeled NPs can be used for cellular monitoring of in vitro cultures and in vivo transplantation. PMID:25304204

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

    PubMed Central

    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.

    PubMed

    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.

    PubMed

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  10. Algal autolysate medium to label proteins for NMR in mammalian cells.

    PubMed

    Fuccio, Carmelo; Luchinat, Enrico; Barbieri, Letizia; Neri, Sara; Fragai, Marco

    2016-04-01

    In-cell NMR provides structural and functional information on proteins directly inside living cells. At present, the high costs of the labeled media for mammalian cells represent a limiting factor for the development of this methodology. Here we report a protocol to prepare a homemade growth medium from Spirulina platensis autolysate, suitable to express uniformly labeled proteins inside mammalian cells at a reduced cost-per-sample. The human proteins SOD1 and Mia40 were overexpressed in human cells grown in (15)N-enriched S. platensis algal-derived medium, and high quality in-cell NMR spectra were obtained. PMID:27106902

  11. Evaluation of red blood cell labelling methods based on a statistical model for red blood cell survival.

    PubMed

    Korell, Julia; Coulter, Carolyn V; Duffull, Stephen B

    2011-12-21

    The aim of this work is to compare different labelling methods that are commonly used to estimate the lifespan of red blood cells (RBCs), e.g. in anaemia of renal failure, where the effect of treatment with erythropoietin depends on the lifespan of RBCs. A previously developed model for the survival time of RBCs that accounts for plausible physiological processes of RBC destruction was used to simulate ideal random and cohort labelling methods for RBCs, as well as the flaws associated with these methods (e.g. reuse of label and loss of the label from the surviving RBCs). Random labelling with radioactive chromium and cohort labelling using heavy nitrogen were considered. Blood sampling times were determined for RBC survival studies using both labelling methods by applying the theory of optimal design. It was assessed whether the underlying parameter values of the model are estimable from these studies, and the precision of the parameter estimates were calculated. In theory, parameter estimation would be possible for both types of ideal labelling methods without flaws. However, flaws associated with random labelling are significant and not all parameters controlling RBC survival in the model can be estimated with good precision. In contrast, cohort labelling shows good precision in the parameter estimates even in the presence of reuse and prolonged incorporation of the label. A model based analysis of RBC survival studies is recommended in future to account for limitations in methodology as well as likely causes of RBC destruction. PMID:21945607

  12. Reconciling Estimates of Cell Proliferation from Stable Isotope Labeling Experiments

    PubMed Central

    Drylewicz, Julia; Elemans, Marjet; Zhang, Yan; Kelly, Elizabeth; Reljic, Rajko; Tesselaar, Kiki; de Boer, Rob J.; Macallan, Derek C.; Borghans, José A. M.; Asquith, Becca

    2015-01-01

    Stable isotope labeling is the state of the art technique for in vivo quantification of lymphocyte kinetics in humans. It has been central to a number of seminal studies, particularly in the context of HIV-1 and leukemia. However, there is a significant discrepancy between lymphocyte proliferation rates estimated in different studies. Notably, deuterated 2H2-glucose (D2-glucose) labeling studies consistently yield higher estimates of proliferation than deuterated water (D2O) labeling studies. This hampers our understanding of immune function and undermines our confidence in this important technique. Whether these differences are caused by fundamental biochemical differences between the two compounds and/or by methodological differences in the studies is unknown. D2-glucose and D2O labeling experiments have never been performed by the same group under the same experimental conditions; consequently a direct comparison of these two techniques has not been possible. We sought to address this problem. We performed both in vitro and murine in vivo labeling experiments using identical protocols with both D2-glucose and D2O. This showed that intrinsic differences between the two compounds do not cause differences in the proliferation rate estimates, but that estimates made using D2-glucose in vivo were susceptible to difficulties in normalization due to highly variable blood glucose enrichment. Analysis of three published human studies made using D2-glucose and D2O confirmed this problem, particularly in the case of short term D2-glucose labeling. Correcting for these inaccuracies in normalization decreased proliferation rate estimates made using D2-glucose and slightly increased estimates made using D2O; thus bringing the estimates from the two methods significantly closer and highlighting the importance of reliable normalization when using this technique. PMID:26437372

  13. Reconciling Estimates of Cell Proliferation from Stable Isotope Labeling Experiments.

    PubMed

    Ahmed, Raya; Westera, Liset; Drylewicz, Julia; Elemans, Marjet; Zhang, Yan; Kelly, Elizabeth; Reljic, Rajko; Tesselaar, Kiki; de Boer, Rob J; Macallan, Derek C; Borghans, José A M; Asquith, Becca

    2015-10-01

    Stable isotope labeling is the state of the art technique for in vivo quantification of lymphocyte kinetics in humans. It has been central to a number of seminal studies, particularly in the context of HIV-1 and leukemia. However, there is a significant discrepancy between lymphocyte proliferation rates estimated in different studies. Notably, deuterated (2)H2-glucose (D2-glucose) labeling studies consistently yield higher estimates of proliferation than deuterated water (D2O) labeling studies. This hampers our understanding of immune function and undermines our confidence in this important technique. Whether these differences are caused by fundamental biochemical differences between the two compounds and/or by methodological differences in the studies is unknown. D2-glucose and D2O labeling experiments have never been performed by the same group under the same experimental conditions; consequently a direct comparison of these two techniques has not been possible. We sought to address this problem. We performed both in vitro and murine in vivo labeling experiments using identical protocols with both D2-glucose and D2O. This showed that intrinsic differences between the two compounds do not cause differences in the proliferation rate estimates, but that estimates made using D2-glucose in vivo were susceptible to difficulties in normalization due to highly variable blood glucose enrichment. Analysis of three published human studies made using D2-glucose and D2O confirmed this problem, particularly in the case of short term D2-glucose labeling. Correcting for these inaccuracies in normalization decreased proliferation rate estimates made using D2-glucose and slightly increased estimates made using D2O; thus bringing the estimates from the two methods significantly closer and highlighting the importance of reliable normalization when using this technique. PMID:26437372

  14. Endowing carbon nanotubes with superparamagnetic properties: applications for cell labeling, MRI cell tracking and magnetic manipulations.

    PubMed

    Lamanna, Giuseppe; Garofalo, Antonio; Popa, Gabriela; Wilhelm, Claire; Bégin-Colin, Sylvie; Felder-Flesch, Delphine; Bianco, Alberto; Gazeau, Florence; Ménard-Moyon, Cécilia

    2013-05-21

    Coating of carbon nanotubes (CNTs) with magnetic nanoparticles (NPs) imparts novel magnetic, optical, and thermal properties with potential applications in the biomedical domain. Multi-walled CNTs have been decorated with iron oxide superparamagnetic NPs. Two different approaches have been investigated based on ligand exchange or "click chemistry". The presence of the NPs on the nanotube surface allows conferring magnetic properties to CNTs. We have evaluated the potential of the NP/CNT hybrids as a contrast agent for magnetic resonance imaging (MRI) and their interactions with cells. The capacity of the hybrids to magnetically monitor and manipulate cells has also been investigated. The NP/CNTs can be manipulated by a remote magnetic field with enhanced contrast in MRI. They are internalized into tumor cells without showing cytotoxicity. The labeled cells can be magnetically manipulated as they display magnetic mobility and are detected at a single cell level through high resolution MRI.

  15. Stable isotope labeling by amino acids in cell culture (SILAC) for quantitative proteomics.

    PubMed

    Hoedt, Esthelle; Zhang, Guoan; Neubert, Thomas A

    2014-01-01

    Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful approach for high-throughput quantitative proteomics. SILAC allows highly accurate protein quantitation through metabolic encoding of whole cell proteomes using stable isotope labeled amino acids. Since its introduction in 2002, SILAC has become increasingly popular. In this chapter we review the methodology and application of SILAC, with an emphasis on three research areas: dynamics of posttranslational modifications, protein-protein interactions, and protein turnover. PMID:24952180

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

    PubMed Central

    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

  17. Cell Labeling for 19F MRI: New and Improved Approach to Perfluorocarbon Nanoemulsion Design

    PubMed Central

    Patel, Sravan K.; Williams, Jonathan; Janjic, Jelena M.

    2013-01-01

    This report describes novel perfluorocarbon (PFC) nanoemulsions designed to improve ex vivo cell labeling for 19F magnetic resonance imaging (MRI). 19F MRI is a powerful non-invasive technique for monitoring cells of the immune system in vivo, where cells are labeled ex vivo with PFC nanoemulsions in cell culture. The quality of 19F MRI is directly affected by the quality of ex vivo PFC cell labeling. When co-cultured with cells for longer periods of time, nanoemulsions tend to settle due to high specific weight of PFC oils (1.5–2.0 g/mL). This in turn can decrease efficacy of excess nanoemulsion removal and reliability of the cell labeling in vitro. To solve this problem, novel PFC nanoemulsions are reported which demonstrate lack of sedimentation and high stability under cell labeling conditions. They are monodisperse, have small droplet size (~130 nm) and low polydispersity (<0.15), show a single peak in the 19F nuclear magnetic resonance spectrum at −71.4 ppm and possess high fluorine content. The droplet size and polydispersity remained unchanged after 160 days of follow up at three temperatures (4, 25 and 37 °C). Further, stressors such as elevated temperature in the presence of cells, and centrifugation, did not affect the nanoemulsion droplet size and polydispersity. Detailed synthetic methodology and in vitro testing for these new PFC nanoemulsions is presented. PMID:25586263

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

    NASA Astrophysics Data System (ADS)

    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

  19. Labeling and Imaging Mesenchymal Stem Cells with Quantum Dots

    EPA Science Inventory

    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...

  20. Cell Proliferation Analysis Using EdU Labeling in Whole Plant and Histological Samples of Arabidopsis.

    PubMed

    Kazda, Anita; Akimcheva, Svetlana; Watson, J Matthew; Riha, Karel

    2016-01-01

    The ability to analyze cell division in both spatial and temporal dimensions within an organism is a key requirement in developmental biology. Specialized cell types within individual organs, such as those within shoot and root apical meristems, have often been identified by differences in their rates of proliferation prior to the characterization of distinguishing molecular markers. Replication-dependent labeling of DNA is a widely used method for assaying cell proliferation. The earliest approaches used radioactive labeling with tritiated thymidine, which were later followed by immunodetection of bromodeoxyuridine (BrdU). A major advance in DNA labeling came with the use of 5-ethynyl-2'deoxyuridine (EdU) which has proven to have multiple advantages over BrdU. Here we describe the methodology for analyzing EdU labeling and retention in whole plants and histological sections of Arabidopsis.

  1. Cell Proliferation Analysis Using EdU Labeling in Whole Plant and Histological Samples of Arabidopsis.

    PubMed

    Kazda, Anita; Akimcheva, Svetlana; Watson, J Matthew; Riha, Karel

    2016-01-01

    The ability to analyze cell division in both spatial and temporal dimensions within an organism is a key requirement in developmental biology. Specialized cell types within individual organs, such as those within shoot and root apical meristems, have often been identified by differences in their rates of proliferation prior to the characterization of distinguishing molecular markers. Replication-dependent labeling of DNA is a widely used method for assaying cell proliferation. The earliest approaches used radioactive labeling with tritiated thymidine, which were later followed by immunodetection of bromodeoxyuridine (BrdU). A major advance in DNA labeling came with the use of 5-ethynyl-2'deoxyuridine (EdU) which has proven to have multiple advantages over BrdU. Here we describe the methodology for analyzing EdU labeling and retention in whole plants and histological sections of Arabidopsis. PMID:26659962

  2. New carboxysilane-coated iron oxide nanoparticles for nonspecific cell labelling.

    PubMed

    Bridot, Jean-Luc; Stanicki, Dimitri; Laurent, Sophie; Boutry, Sébastien; Gossuin, Yves; Leclère, Philippe; Lazzaroni, Roberto; Vander Elst, Luce; Muller, Robert N

    2013-01-01

    Magnetic resonance imaging (MRI) offers the possibility of tracking cells labelled with a contrast agent and evaluating the progress of cell therapies. This requires efficient cell labelling with contrast agents. A basic incubation of cells with iron oxide nanoparticles (NPs) is a common method. This study reports the synthesis at the gram scale of iron oxide nanoparticles as MRI T₂ contrast agents for cell labelling. These NPs are based on small iron oxide cores coated with a thin polysiloxane shell presenting carboxylic acid functions. The iron oxide cores produced have been characterized by transmission electron microscopy, X-ray diffraction, ζ-potential, infrared, photon correlation spectroscopy, atomic force microscopy, magnetometry and relaxometric measurements. These measurements confirmed the expected surface modification by carboxysilane. Carboxylic groups created electrostatic repulsion between NPs when they are deprotonated. Therefore, highly concentrated aqueous solutions of carboxysilane coated iron oxide NPs can be obtained, up to 70% (w/w). These NPs could be used for cell labelling owing to their aggregation and re-dispersion properties. NPs precipitated in Dulbecco's modified Eagle medium induced a rapid association with 3 T6 fibroblast cells and could easily be re-dispersed in phosphate buffer saline solution to obtain properly labelled cells. PMID:24375902

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

    PubMed

    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.

  4. Noninvasive Tracking of Encapsulated Insulin Producing Cells Labelled with Magnetic Microspheres by Magnetic Resonance Imaging

    PubMed Central

    Yim, Mandy M. W.; Foster, Jayne L.; Oberholzer, Jose

    2016-01-01

    Microencapsulated islets are usually injected free-floating into the peritoneal cavity, so the position of the grafts remains elusive after transplantation. This study aims to assess magnetic resonance imaging (MRI) as a noninvasive means to track microencapsulated insulin producing cells following transplantation. Encapsulated insulin producing cells (MIN6 and human islets) were labelled with magnetic microspheres (MM), assessed for viability and insulin secretion, and imaged in vitro using a clinical grade 3 T MRI and in vivo using both clinical grade 3 T and research grade 11.7 T MRI. Fluorescent imaging demonstrated the uptake of MM by both MIN6 and human islets with no changes in cell morphology and viability. MM labelling did not affect the glucose responsiveness of encapsulated MIN6 and islets in vitro. In vivo encapsulated MM-labelled MIN6 normalized sugar levels when transplanted into diabetic mice. In vitro MRI demonstrated that single microcapsules as well as clusters of encapsulated MM-labelled cells could be visualised clearly in agarose gel phantoms. In vivo encapsulated MM-labelled MIN6 could be visualised more clearly within the peritoneal cavity as discrete hypointensities using the high power 11.7 T but not the clinical grade 3 T MRI. This study demonstrates a method to noninvasively track encapsulated insulin producing cells by MM labelling and MRI.

  5. Noninvasive Tracking of Encapsulated Insulin Producing Cells Labelled with Magnetic Microspheres by Magnetic Resonance Imaging

    PubMed Central

    Yim, Mandy M. W.; Foster, Jayne L.; Oberholzer, Jose

    2016-01-01

    Microencapsulated islets are usually injected free-floating into the peritoneal cavity, so the position of the grafts remains elusive after transplantation. This study aims to assess magnetic resonance imaging (MRI) as a noninvasive means to track microencapsulated insulin producing cells following transplantation. Encapsulated insulin producing cells (MIN6 and human islets) were labelled with magnetic microspheres (MM), assessed for viability and insulin secretion, and imaged in vitro using a clinical grade 3 T MRI and in vivo using both clinical grade 3 T and research grade 11.7 T MRI. Fluorescent imaging demonstrated the uptake of MM by both MIN6 and human islets with no changes in cell morphology and viability. MM labelling did not affect the glucose responsiveness of encapsulated MIN6 and islets in vitro. In vivo encapsulated MM-labelled MIN6 normalized sugar levels when transplanted into diabetic mice. In vitro MRI demonstrated that single microcapsules as well as clusters of encapsulated MM-labelled cells could be visualised clearly in agarose gel phantoms. In vivo encapsulated MM-labelled MIN6 could be visualised more clearly within the peritoneal cavity as discrete hypointensities using the high power 11.7 T but not the clinical grade 3 T MRI. This study demonstrates a method to noninvasively track encapsulated insulin producing cells by MM labelling and MRI. PMID:27631014

  6. Iron oxide labelling of human mesenchymal stem cells in collagen hydrogels for articular cartilage repair.

    PubMed

    Heymer, Andrea; Haddad, Daniel; Weber, Meike; Gbureck, Uwe; Jakob, Peter M; Eulert, Jochen; Nöth, Ulrich

    2008-04-01

    For the development of new therapeutical cell-based strategies for articular cartilage repair, a reliable cell monitoring technique is required to track the cells in vivo non-invasively and repeatedly. We present a systematic and detailed study on the performance and biological impact of a simple and efficient labelling protocol for human mesenchymal stem cells (hMSCs). Commercially available very small superparamagnetic iron oxide particles (VSOPs) were used as magnetic resonance (MR) contrast agent. Iron uptake via endocytosis was confirmed histologically with prussian blue staining and quantified by mass spectrometry. Compared with unlabelled cells, VSOP-labelling did neither influence the viability nor the proliferation potential of hMSCs. Furthermore, iron incorporation did not affect hMSCs in undergoing adipogenic, osteogenic or chondrogenic differentiation, as demonstrated histologically and by gene expression analyses. The efficiency of the labelling protocol was assessed with high-resolution MR imaging at 11.7T. VSOP-labelled hMSCs were visualised in a collagen type I hydrogel, which is in clinical use for matrix-based articular cartilage repair. The presence of VSOP-labelled hMSCs was indicated by distinct hypointense spots in the MR images, as a result of iron specific loss of signal intensity. In summary, this labelling technique has great potential to visualise hMSCs and track their migration after transplantation for articular cartilage repair with MR imaging.

  7. Noninvasive Tracking of Encapsulated Insulin Producing Cells Labelled with Magnetic Microspheres by Magnetic Resonance Imaging.

    PubMed

    Vaithilingam, Vijayaganapathy; Yim, Mandy M W; Foster, Jayne L; Stait-Gardner, Timothy; Oberholzer, Jose; Tuch, Bernard E

    2016-01-01

    Microencapsulated islets are usually injected free-floating into the peritoneal cavity, so the position of the grafts remains elusive after transplantation. This study aims to assess magnetic resonance imaging (MRI) as a noninvasive means to track microencapsulated insulin producing cells following transplantation. Encapsulated insulin producing cells (MIN6 and human islets) were labelled with magnetic microspheres (MM), assessed for viability and insulin secretion, and imaged in vitro using a clinical grade 3 T MRI and in vivo using both clinical grade 3 T and research grade 11.7 T MRI. Fluorescent imaging demonstrated the uptake of MM by both MIN6 and human islets with no changes in cell morphology and viability. MM labelling did not affect the glucose responsiveness of encapsulated MIN6 and islets in vitro. In vivo encapsulated MM-labelled MIN6 normalized sugar levels when transplanted into diabetic mice. In vitro MRI demonstrated that single microcapsules as well as clusters of encapsulated MM-labelled cells could be visualised clearly in agarose gel phantoms. In vivo encapsulated MM-labelled MIN6 could be visualised more clearly within the peritoneal cavity as discrete hypointensities using the high power 11.7 T but not the clinical grade 3 T MRI. This study demonstrates a method to noninvasively track encapsulated insulin producing cells by MM labelling and MRI. PMID:27631014

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

    NASA Astrophysics Data System (ADS)

    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.

  9. A Dual SILAC Proteomic Labeling Strategy for Quantifying Constitutive and Cell-Cell Induced Protein Secretion.

    PubMed

    Stiess, Michael; Wegehingel, Sabine; Nguyen, Chuong; Nickel, Walter; Bradke, Frank; Cambridge, Sidney B

    2015-08-01

    Recent evidence suggests that the extracellular protein milieu is much more complex than previously assumed as various secretome analyses from different cell types described the release of hundreds to thousands of proteins. The extracellular function of many of these proteins has yet to be determined particularly in the context of three-dimensional tissues with abundant cell-cell contacts. Toward this goal, we developed a strategy of dual SILAC labeling astrocytic cultures for in silico exclusion of unlabeled proteins from serum or neurons used for stimulation. For constitutive secretion, this strategy allowed the precise quantification of the extra-to-intracellular protein ratio of more than 2000 identified proteins. Ratios covered 4 orders of magnitude indicating that the intracellular vs extracellular contributions of different proteins can be variable. Functionally, the secretome of labeled forebrain astrocytic cultures specifically changed within hours after adding unlabeled, "physiological" forebrain neurons. "Nonphysiological" cerebellar hindbrain neurons, however, elicited a different, highly repulsive secretory response. Our data also suggest a significant association of constitutive secretion with the classical secretion pathway and regulated secretion with unconventional pathways. We conclude that quantitative proteomics can help to elucidate general principles of cellular secretion and provide functional insight into the abundant extracellular presence of proteins.

  10. Accuracy of blood volume estimations in critically ill children using 125I-labelled albumin and 51Cr-labelled red cells.

    PubMed

    Linderkamp, O; Holthausen, H; Seifert, J; Butenandt, I; Riegel, K P

    1977-06-01

    Blood volume was estimated using 51chromium labelled red cells and 125iodinated human serum albumin in 5 children with sepsis, in 6 burned children and 7 children with acute lymphoblastic leukaemia. Studies of the equilibration pattern demonstrated that the mixing time of labelled red cells was prolonged to 40 minutes or more in 5 children, indicating the existence of slowly circulating red cells. Mixing of labelled albumin was complete within 10 minutes in 15 patients and within 20 minutes in all the children studied. In a burned patient with severe sepsis, exchange transfusion improved the clinical state and normalized the equilibration pattern of labelled red cells. The mean body/venous haematocrit ratio was 0.893+/-0.018 (SD) in the children with sepsis, 0.859+/-0.052 in the burned patients, and 0.916+/-0.078 in the children with acute lymphoblastic leukaemia, increasing with spleen size in the latter group. PMID:267010

  11. Selective Methyl Labeling of Eukaryotic Membrane Proteins Using Cell-Free Expression

    PubMed Central

    2015-01-01

    Structural characterization of membrane proteins and other large proteins with NMR relies increasingly on perdeuteration combined with incorporation of specifically protonated amino acid moieties, such as methyl groups of isoleucines, valines, or leucines. The resulting proton dilution reduces dipolar broadening producing sharper resonance lines, ameliorates spectral crowding, and enables measuring of crucial distances between and to methyl groups. While incorporation of specific methyl labeling is now well established for bacterial expression using suitable precursors, corresponding methods are still lacking for cell-free expression, which is often the only choice for producing labeled eukaryotic membrane proteins in mg quantities. Here we show that we can express methyl-labeled human integral membrane proteins cost-effectively by cell-free expression based of crude hydrolyzed ILV-labeled OmpX inclusion bodies. These are obtained in Escherichia coli with very high quantity and represent an optimal intermediate to channel ILV precursors into the eukaryotic proteins. PMID:24937763

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

    SciTech Connect

    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.

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

    PubMed Central

    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

  14. Acoustofluidic, label-free separation and simultaneous concentration of rare tumor cells from white blood cells.

    PubMed

    Antfolk, Maria; Magnusson, Cecilia; Augustsson, Per; Lilja, Hans; Laurell, Thomas

    2015-09-15

    Enrichment of rare cells from peripheral blood has emerged as a means to enable noninvasive diagnostics and development of personalized drugs, commonly associated with a prerequisite to concentrate the enriched rare cell population prior to molecular analysis or culture. However, common concentration by centrifugation has important limitations when processing low cell numbers. Here, we report on an integrated acoustophoresis-based rare cell enrichment system combined with integrated concentration. Polystyrene 7 μm microparticles could be separated from 5 μm particles with a recovery of 99.3 ± 0.3% at a contamination of 0.1 ± 0.03%, with an overall 25.7 ± 1.7-fold concentration of the recovered 7 μm particles. At a flow rate of 100 μL/min, breast cancer cells (MCF7) spiked into red blood cell-lysed human blood were separated with an efficiency of 91.8 ± 1.0% with a contamination of 0.6 ± 0.1% from white blood cells with a 23.8 ± 1.3-fold concentration of cancer cells. The recovery of prostate cancer cells (DU145) spiked into whole blood was 84.1 ± 2.1% with 0.2 ± 0.04% contamination of white blood cells with a 9.6 ± 0.4-fold concentration of cancer cells. This simultaneous on-chip separation and concentration shows feasibility of future acoustofluidic systems for rapid label-free enrichment and molecular characterization of circulating tumor cells using peripheral venous blood in clinical practice.

  15. Affinity labeling and binding of nitrobenzylthionosine (NBTI) to a membrane fraction (MF) of cultured cell lines

    SciTech Connect

    Woffendin, C.; Plagemann, P.G.W.

    1986-05-01

    Equilibrium binding identified high affinity NBTI binding sites (K/sub D/ = 1-3 nM) on the MF's of L929, L1210, P388, S49 and CHO cells. High affinity NBTI binding sites are associated with the nucleoside transporter since none were present in a MF of a transport-deficient mutant of S49 cells (AE1). MF's of Novikoff cells, like intact Novikoff cells, also lacked high affinity NBTI binding sites. MF's of the cell lines were equilibrium labeled with (/sup 3/H)NBTI using photoaffinity conditions and analyzed by SDS-polyacrylamide gel electrophoresis. Radioactivity was specifically incorporated covalently into a 50-70 Kd protein fraction, but the labeled proteins from CHO and L929 cells had a higher apparent molecular weight than those from S49 and P388 cells. In addition, in MF's from some cell lines lower molecular weight components became photoaffinity labeled. Maximum photoaffinity labeling of the MF proteins was observed with much higher (/sup 3/H)NBTI concentrations (100-200 nM) than those saturating the nucleoside transporter. This finding is explained by a reduced affinity of the photoactivated NBTI intermediate(s) for the transporter. When detergent solubilized MF's from cultured cells were chromotographed on a DEAE cellulose column, only 5-10% of the protein, but practically all high affinity NBTI sites, were recovered in the flow through fraction.

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

    NASA Astrophysics Data System (ADS)

    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.

  17. EdU, a new thymidine analogue for labelling proliferating cells in the nervous system.

    PubMed

    Chehrehasa, Fatemah; Meedeniya, Adrian C B; Dwyer, Patrick; Abrahamsen, Greger; Mackay-Sim, Alan

    2009-02-15

    Labelling and identifying proliferating cells is central to understanding neurogenesis and neural lineages in vivo and in vitro. We present here a novel thymidine analogue, ethynyl deoxyuridine (EdU) for labelling dividing cells, detected with a fluorescent azide which forms a covalent bond via the "click" chemistry reaction (the Huisgen 1,3-dipolar cycloaddition reaction of an organic azide to a terminal acetylene). Unlike the commonly used BrdU, EdU detection requires no heat or acid treatment. It is quick and easy and compatible with multiple probes for fluorescence immunochemistry, facilitating the characterisation of proliferating cells at high resolution.

  18. Labeling Cell Surface GPIs and GPI-Anchored Proteins through Metabolic Engineering with Artificial Inositol Derivatives.

    PubMed

    Lu, Lili; Gao, Jian; Guo, Zhongwu

    2015-08-10

    Glycosylphosphatidylinositol (GPI) anchoring of proteins to the cell surface is important for various biological processes, but GPI-anchored proteins are difficult to study. An effective strategy was developed for the metabolic engineering of cell-surface GPIs and GPI-anchored proteins by using inositol derivatives carrying an azido group. The azide-labeled GPIs and GPI-anchored proteins were then tagged with biotin on live cells through a click reaction, which allows further elaboration with streptavidin-conjugated dyes or other molecules. The strategy can be used to label GPI-anchored proteins with various tags for biological studies.

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

    PubMed

    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

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

    NASA Astrophysics Data System (ADS)

    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

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

    PubMed Central

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

    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 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 can be imaged in

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27174199

  4. Assessment of a Nuclear Affinity Labeling Method for Tracking Implanted Mesenchymal Stem Cells

    PubMed Central

    Leiker, Merced; Suzuki, Gen; Iyer, Vijay S.; Canty, John M.; Lee, Techung

    2010-01-01

    Therapeutic implantation of mesenchymal stem cells (MSCs) is entering the realm of clinical trials for several human diseases, and yet much remains uncertain regarding their dynamic distribution and cell fate after in vivo application. Discrepancies in the literature can be attributed in part to the use of different cell labeling/tracking methods and cell administration protocols. To identify a stem cell detection method suitable for myocardial implantation in a large animal model, we experimented on three different MSC labeling methods: adenovirus-mediated expression of enhanced green fluorescence protein (EGFP) and β-galactosidase (LacZ), and nuclear staining with DAPI. Intramuscular and intracoronary administrations of labeled porcine MSCs identified the nuclear affinity dye to be a reliable stem cell tracking marker. Stem cell identification is facilitated by an optimized live cell labeling condition generating bright blue fluorescence sharply confined to the nucleus. DAPI-labeled MSCs retained full viability, ceased proliferation, and exhibited an increased differentiation potential. The labeled MSCs remained fully active in expressing key growth factor and cytokine genes, and notably exhibited enhanced expression of the chemokine receptor CXCR4 and its ligand SDF1, indicating their competency in response to tissue injury. Histological analysis revealed that approximately half a million MSCs or ∼2% of the administered MSCs remained localized in the normal pig heart 2 weeks after coronary infusion. That the vast majority of these identified MSCs were interstitial indicated the ability of MSCs to migrate across the coronary endothelium. No evidence was obtained indicating MSC differentiation to cardiomyocyte. PMID:19069634

  5. Uncovering stem-cell heterogeneity in the microniche with label-free microfluidics

    NASA Astrophysics Data System (ADS)

    Sohn, Lydia L.

    2013-03-01

    Better suited for large number of cells from bulk tissue, traditional cell-screening techniques, such as fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS), cannot easily screen stem or progenitor cells from minute populations found in their physiological niches. Furthermore, they rely upon irreversible antibody binding, potentially altering cell properties, including gene expression and regenerative capacity. We have developed a label-free, single-cell analysis microfluidic platform capable of quantifying cell-surface marker expression of functional organ stem cells directly isolated from their micro-anatomical niche. With this platform, we have screened single quiescent muscle stem (satellite) cells derived from single myofibers, and we have uncovered an important heterogeneity in the surface-marker expression of these cells. By sorting the screened cells with our microfluidic device, we have determined what this heterogeneity means in terms of muscle stem-cell functionality. For instance, we show that the levels of beta1-integrin can predict the differentiation capacity of quiescent satellite cells, and in contrast to recent literature, that some CXCR4 + cells are not myogenic. Our results provide the first direct demonstration of a microniche-specific variation in gene expression in stem cells of the same lineage. Overall, our label-free, single-cell analysis and cell-sorting platform could be extended to other systems involving rare-cell subsets. This work was funded by the W. M. Keck Foundation, NIH, and California Institute of Regenerative Medicine

  6. Kit for the selective labeling of red blood cells in whole blood with [sup 99]Tc

    DOEpatents

    Srivastava, S.C.; Babich, J.W.; Straub, R.; Richards, P.

    1992-05-26

    Disclosed herein are a method and kit for the preparation of [sup 99m]Tc labeled red blood cells using whole blood in a closed sterile system containing stannous tin in a form such that it will enter the red blood cells and be available therein for reduction of technetium. No Drawings

  7. Kit for the selective labeling of red blood cells in whole blood with .sup.9 TC

    DOEpatents

    Srivastava, Suresh C.; Babich, John W.; Straub, Rita; Richards, Powell

    1992-01-01

    Disclosed herein are a method and kit for the preparation of .sup.99m Tc labeled red blood cells using whole blood in a closed sterile system containing stannous tin in a form such that it will enter the red blood cells and be available therein for reduction of technetium.

  8. Single-cell printer: automated, on demand, and label free.

    PubMed

    Gross, Andre; Schöndube, Jonas; Niekrawitz, Sonja; Streule, Wolfgang; Riegger, Lutz; Zengerle, Roland; Koltay, Peter

    2013-12-01

    Within the past years, single-cell analysis has developed into a key topic in cell biology to study cellular functions that are not accessible by investigation of larger cell populations. Engineering approaches aiming to access single cells to extract information about their physiology, phenotype, and genotype at the single-cell level are going manifold ways, meanwhile allowing separation, sorting, culturing, and analysis of individual cells. Based on our earlier research toward inkjet-like printing of single cells, this article presents further characterization results obtained with a fully automated prototype instrument for printing of single living cells in a noncontact inkjet-like manner. The presented technology is based on a transparent microfluidic drop-on-demand dispenser chip coupled with a camera-assisted automatic detection system. Cells inside the chip are detected and classified with this detection system before they are expelled from the nozzle confined in microdroplets, thus enabling a "one cell per droplet" printing mode. To demonstrate the prototype instrument's suitability for biological and biomedical applications, basic experiments such as printing of single-bead and cell arrays as well as deposition and culture of single cells in microwell plates are presented. Printing efficiencies greater than 80% and viability rates about 90% were achieved.

  9. Quantum dot labeling and tracking of cultured limbal epithelial cell transplants in-vitro

    PubMed Central

    Genicio, Nuria; Paramo, Juan Gallo; Shortt, Alex J.

    2015-01-01

    PURPOSE Cultured human limbal epithelial cells (HLEC) have shown promise in the treatment of limbal stem cell deficiency but little is known about their survival, behaviour and long-term fate post transplantation. The aim of this research was to evaluate, in-vitro, quantum dot (QDot) technology as a tool for tracking transplanted HLEC. METHODS In-vitro cultured HLEC were labeled with Qdot nanocrystals. Toxicity was assessed using live-dead assays. The effect on HLEC function was assessed using colony forming efficiency assays and expression of CK3, P63alpha and ABCG2. Sheets of cultured HLEC labeled with Qdot nanocrystals were transplanted onto decellularised human corneo-scleral rims in an organ culture model and observed to investigate the behaviour of transplanted cells. RESULTS Qdot labeling had no detrimental effect on HLEC viability or function in-vitro. Proliferation resulted in a gradual reduction in Qdot signal but sufficient signal was present to allow tracking of cells through multiple generations. Cells labeled with Qdots could be reliably detected and observed using confocal microscopy for at least 2 weeks post transplantation in our organ culture model. In addition it was possible to label and observe epithelial cells in intact human corneas using the Rostock corneal module adapted for use with the Heidelberg HRA. CONCLUSIONS This work demonstrates that Qdots combined with existing clinical equipment could be used to track HLEC for up to 2 weeks post transplantation, however, our model does not permit the assessment of cell labeling beyond 2 weeks. Further characterisation in in-vivo models are required. PMID:26024089

  10. Escherichia coli cell-free protein synthesis and isotope labeling of mammalian proteins.

    PubMed

    Terada, Takaho; Yokoyama, Shigeyuki

    2015-01-01

    This chapter describes the cell-free protein synthesis method, using an Escherichia coli cell extract. This is a cost-effective method for milligram-scale protein production and is particularly useful for the production of mammalian proteins, protein complexes, and membrane proteins that are difficult to synthesize by recombinant expression methods, using E. coli and eukaryotic cells. By adjusting the conditions of the cell-free method, zinc-binding proteins, disulfide-bonded proteins, ligand-bound proteins, etc., may also be produced. Stable isotope labeling of proteins can be accomplished by the cell-free method, simply by using stable isotope-labeled amino acid(s) in the cell-free reaction. Moreover, the cell-free protein synthesis method facilitates the avoidance of stable isotope scrambling and dilution over the recombinant expression methods and is therefore advantageous for amino acid-selective stable isotope labeling. Site-specific stable isotope labeling is also possible with a tRNA molecule specific to the UAG codon. By the cell-free protein synthesis method, coupled transcription-translation is performed from a plasmid vector or a PCR-amplified DNA fragment encoding the protein. A milligram quantity of protein can be produced with a milliliter-scale reaction solution in the dialysis mode. More than a thousand solution structures have been determined by NMR spectroscopy for uniformly labeled samples of human and mouse functional domain proteins, produced by the cell-free method. Here, we describe the practical aspects of mammalian protein production by the cell-free method for NMR spectroscopy.

  11. Advances in chemical labeling of proteins in living cells

    PubMed Central

    Yan, Qi; Bruchez, Marcel P.

    2015-01-01

    Summary The pursuit of quantitative biological information with imaging requires robust labeling approaches that can be used in multiple applications and with a variety of detectable colors and properties. In addition to conventional fluorescent proteins, chemists and biologists have come together to provide a range of approaches that combine dye chemistry with the convenience of genetic targeting. This hybrid-tagging approach combines the rational design of properties available through synthetic dye chemistry with the robust biological targeting available with genetic encoding. In this review, we discuss the current range of approaches that have been exploited for dye targeting, or targeting and activation, and some of the recent applications that are uniquely enabled by these hybrid-tagging approaches. PMID:25743694

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

    PubMed

    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

  13. From seeing to believing: labelling strategies for in vivo cell-tracking experiments.

    PubMed

    Progatzky, Fränze; Dallman, Margaret J; Lo Celso, Cristina

    2013-06-01

    Intravital microscopy has become increasingly popular over the past few decades because it provides high-resolution and real-time information about complex biological processes. Technological advances that allow deeper penetration in live tissues, such as the development of confocal and two-photon microscopy, together with the generation of ever-new fluorophores that facilitate bright labelling of cells and tissue components have made imaging of vertebrate model organisms efficient and highly informative. Genetic manipulation leading to expression of fluorescent proteins is undoubtedly the labelling method of choice and has been used to visualize several cell types in vivo. This approach, however, can be technically challenging and time consuming. Over the years, several dyes have been developed to allow rapid, effective and bright ex vivo labelling of cells for subsequent transplantation and imaging. Here, we review and discuss the advantages and limitations of a number of strategies commonly used to label and track cells at high resolution in vivo in mouse and zebrafish, using fluorescence microscopy. While the quest for the perfect label is far from achieved, current reagents are valuable tools enabling the progress of biological discovery, so long as they are selected and used appropriately.

  14. Illumination of growth, division and secretion by metabolic labeling of the bacterial cell surface

    PubMed Central

    Siegrist, M. Sloan; Swarts, Benjamin M.; Fox, Douglas M.; Lim, Shion An; Bertozzi, Carolyn R.

    2015-01-01

    The cell surface is the essential interface between a bacterium and its surroundings. Composed primarily of molecules that are not directly genetically encoded, this highly dynamic structure accommodates the basic cellular processes of growth and division as well as the transport of molecules between the cytoplasm and the extracellular milieu. In this review, we describe aspects of bacterial growth, division and secretion that have recently been uncovered by metabolic labeling of the cell envelope. Metabolite derivatives can be used to label a variety of macromolecules, from proteins to non-genetically-encoded glycans and lipids. The embedded metabolite enables precise tracking in time and space, and the versatility of newer chemoselective detection methods offers the ability to execute multiple experiments concurrently. In addition to reviewing the discoveries enabled by metabolic labeling of the bacterial cell envelope, we also discuss the potential of these techniques for translational applications. Finally, we offer some guidelines for implementing this emerging technology. PMID:25725012

  15. Label-free multiphoton imaging and photoablation of preinvasive cancer cells

    NASA Astrophysics Data System (ADS)

    Zhuo, Shuangmu; Chen, Jianxin; Wu, Guizhu; Zhu, Xiaoqin; Jiang, Xingshan; Xie, Shusen

    2012-01-01

    Detection and treatment of early lesions in epithelial tissue offer several possibilities for curing cancer, but it is challenging. Here, we present an optical technique, the combination of multiphoton imaging and absorption, to label-freely detect and ablate preinvasive cancer cells in epithelial tissue. We find that multiphoton imaging can label-freely visualize the principal features of nuclear atypia associated with epithelial precancerous lesions, and the spatial localization of multiphoton absorption can perform targeted ablation of preinvasive cancer cells with micrometer-sized volume precision. These results indicate that this optical technique has the capability to label-freely visualize and remove preinvasive cancer cells in epithelial tissue. This study highlights the potential of this technique as a "seek-and-treat" tool for early lesions in epithelial tissue.

  16. Freeze-fracture cytochemistry: thin sections of cells and tissues after labeling of fractures faces.

    PubMed

    da Silva, P P; Parkison, C; Dwyer, N

    1981-08-01

    Experimental details of a new method for the cytochemical characterization of the membrane faces and cytoplasm produced by freeze-fracture of isolated cells and tissues are presented. This new method-"fracture-label"-involves grinding of frozen samples immersed in liquid nitrogen, thawing, cytochemical labeling of the fractured faces, and processing for thin section electron microscopy. Cationized ferritin (at pH. 7.5 and 4.0), colloidal iron, as well as concanavalin A are used to label the fractures faces of leukocytes and Hela cells embedded in a cross-linked matrix of bovine serum albumin and of liver and spleen tissues. Our results show the presence of numerous anionic binding sites on the fracture faces of all plasma and cytoplasmic membranes, and of concanavalin A binding sites preferentially associated to the exoplasmic fracture faces of plasma and nuclear envelope membranes. A proportion of the anionic sites appears to be revealed by, or during, the freeze-fracture process. Colloidal iron labeling also shows preferential association with the chromatin areas of cross-fractured nuclei. The results show that "fracture-label", i.e., the combined application of freeze-fracture and cytochemical labeling techniques, can be used to study the surface chemistry of the fractures faces of biological membranes as well as of cross-fractured cytoplasm. PMID:7276536

  17. 40 CFR 600.304-12 - Fuel economy label-special requirements for hydrogen fuel cell vehicles.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 31 2013-07-01 2013-07-01 false Fuel economy label-special requirements for hydrogen fuel cell vehicles. 600.304-12 Section 600.304-12 Protection of Environment... fuel cell vehicles. Fuel economy labels for hydrogen fuel cell vehicles must meet the...

  18. 40 CFR 600.304-12 - Fuel economy label-special requirements for hydrogen fuel cell vehicles.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 31 2012-07-01 2012-07-01 false Fuel economy label-special requirements for hydrogen fuel cell vehicles. 600.304-12 Section 600.304-12 Protection of Environment... fuel cell vehicles. Fuel economy labels for hydrogen fuel cell vehicles must meet the...

  19. 40 CFR 600.304-12 - Fuel economy label-special requirements for hydrogen fuel cell vehicles.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 30 2014-07-01 2014-07-01 false Fuel economy label-special requirements for hydrogen fuel cell vehicles. 600.304-12 Section 600.304-12 Protection of Environment... fuel cell vehicles. Fuel economy labels for hydrogen fuel cell vehicles must meet the...

  20. [Investigation of 5-bromo-2'-deoxyuridine labelling mice retinal progenitor cells].

    PubMed

    Sun, Xuerong; Dong, Zhizhang; Deng, Fei; Hu, Huiling; Ge, Jian

    2013-02-01

    BrdU (5-Bromo-2'-deoxyuridine) is usually used to label the mitotic cells as well as to trace reagent in cell transplation. However, BrdU could also exert some side effect on cellular biological characteristics upon inappropriate use. To explore the appropriate concentration of BrdU for labelling retinal progenitor cells (RPCs), we co-cultured Embryonic day (E) 17. 5 RPCs with different concentrations of BrdU, which were 0.2, 1, 5 and 10 micromol/L, respectively. After 48 hours, the RPCs were proliferation- or differentiation-cultured. Immunofluorescence was used to detect the BrdU-positive ratio and differentiation potential. Cell count was used to evaluate proliferation ability, and lactate dehydrogenase (LDH) release assay was used to monitor cytotoxicity. The results showed that 0.2 micromol/ L BrdU could not label RPCs clearly, while BrdU of 1, 5 or 10 micromol/L could label the RPCs with similar ratios. 1 micromol/L BrdU displayed no obvious cytotoxicity and showed no obvious effect on the proliferation and differentiation ability. However, 5 micromol/L or 10 micromol/L BrdU could evidently inhibit RPCs proliferation, partly due to the cytotoxicity effect. Furthermore, 10 micromol/L BrdU could inhibit the differentiation of RPCs towards MAP2-positive nerve cells, but showed no influence on the differentiation of RPCs towards GFAP- and glutamine synthetase positive glial cells. This study suggested that 1 micromol/L BrdU could be an appropriate concentration for RPCs labelling and could efficiently label RPCs without obvious side effect. PMID:23488152

  1. Magnetic resonance investigation of magnetic-labeled baker's yeast cells

    NASA Astrophysics Data System (ADS)

    Godoy Morais, J. P. M.; Azevedo, R. B.; Silva, L. P.; Lacava, Z. G. M.; Báo, S. N.; Silva, O.; Pelegrini, F.; Gansau, C.; Buske, N.; Safarik, I.; Safarikova, M.; Morais, P. C.

    2004-05-01

    In this study, the interaction of DMSA-coated magnetite nanoparticles (5 and 10 nm core-size) with Saccharomyces cerevisae was investigated using magnetic resonance (MR) and transmission electron microscopy (TEM). The TEM micrographs revealed magnetite nanoparticles attached externally to the cell wall. The MR data support the strong interaction among the nanoparticles supported by the cells. A remarkable shift in the resonance field was used as signature of particle attachment to the cell wall.

  2. A chronic 1 year assessment of MRI contrast agent-labelled neural stem cell transplants in stroke.

    PubMed

    Modo, M; Beech, J S; Meade, T J; Williams, S C R; Price, J

    2009-08-01

    Non-invasive identification of transplanted neural stem cells in vivo by pre-labelling with contrast agents may play an important role in the translation of cell therapy to the clinic. Understanding the impact of these labels on the cells' ability to repair is therefore vital. In rats with middle cerebral artery occlusion (MCAo), a model of stroke, the transhemispheric migration of MHP36 cells labelled with the bimodal contrast agent GRID was detected on magnetic resonance images (MRI) up to 4 weeks following transplantation. However, compared to MHP36 cells labelled with the red fluorescent dye PKH26, GRID-labelled transplants did not significantly improve behaviour, and performance was akin to non-treated animals. Likewise, the evolution of anatomical damage as assessed by serial, T(2)-weighted MRI over 1 year indicated that GRID-labelled transplants resulted in a slight increase in lesion size compared to MCAo-only animals, whereas the same, PKH26-labelled cells significantly decreased lesion size by 35%. Although GRID labelling allows the in vivo identification of transplanted cells up to 1 month after transplantation, it is likely that some is gradually degraded inside cells. The translation of cellular imaging therefore does not only require the in vitro assessment of contrast agents on cellular functions, but also requires the chronic, in vivo assessment of the label on the stem cells' ability to repair in preclinical models of neurological disease. PMID:18634886

  3. Tracking Transplanted Stem Cells Using Magnetic Resonance Imaging and the Nanoparticle Labeling Method in Urology

    PubMed Central

    Kim, Jae Heon; Lee, Hong J.; Song, Yun Seob

    2015-01-01

    A reliable in vivo imaging method to localize transplanted cells and monitor their viability would enable a systematic investigation of cell therapy. Most stem cell transplantation studies have used immunohistological staining, which does not provide information about the migration of transplanted cells in vivo in the same host. Molecular imaging visualizes targeted cells in a living host, which enables determining the biological processes occurring in transplanted stem cells. Molecular imaging with labeled nanoparticles provides the opportunity to monitor transplanted cells noninvasively without sacrifice and to repeatedly evaluate them. Among several molecular imaging techniques, magnetic resonance imaging (MRI) provides high resolution and sensitivity of transplanted cells. MRI is a powerful noninvasive imaging modality with excellent image resolution for studying cellular dynamics. Several types of nanoparticles including superparamagnetic iron oxide nanoparticles and magnetic nanoparticles have been used to magnetically label stem cells and monitor viability by MRI in the urologic field. This review focuses on the current role and limitations of MRI with labeled nanoparticles for tracking transplanted stem cells in urology. PMID:26413510

  4. Label-Free Segmentation of Co-cultured Cells on a Nanotopographical Gradient

    PubMed Central

    2012-01-01

    The function and fate of cells is influenced by many different factors, one of which is surface topography of the support culture substrate. Systematic studies of nanotopography and cell response have typically been limited to single cell types and a small set of topographical variations. Here, we show a radical expansion of experimental throughput using automated detection, measurement, and classification of co-cultured cells on a nanopillar array where feature height changes continuously from planar to 250 nm over 9 mm. Individual cells are identified and characterized by more than 200 descriptors, which are used to construct a set of rules for label-free segmentation into individual cell types. Using this approach we can achieve label-free segmentation with 84% confidence across large image data sets and suggest optimized surface parameters for nanostructuring of implant devices such as vascular stents. PMID:23252684

  5. Magnetic Cell Labeling of Primary and Stem Cell-Derived Pig Hepatocytes for MRI-Based Cell Tracking of Hepatocyte Transplantation

    PubMed Central

    Roach, Dwayne R.; Garrett, Wesley M.; Welch, Glenn; Caperna, Thomas J.; Talbot, Neil C.; Shapiro, Erik M.

    2015-01-01

    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 work, we describe culture conditions for magnetic cell labeling of cells from two different pig hepatocyte cell sources; primary pig hepatocytes (ppHEP) and stem cell-derived hepatocytes (PICM-19FF). The magnetic particle is a micron-sized iron oxide particle (MPIO) that has been extensively studied for magnetic cell labeling for MRI-based cell tracking. ppHEP could endocytose MPIO with labeling percentages as high as 70%, achieving iron content as high as ~55 pg/cell, with >75% viability. PICM-19FF had labeling >97%, achieving iron content ~38 pg/cell, with viability >99%. Extensive morphological and functional assays indicated that magnetic cell labeling was benign to the cells. The results encourage the use of MRI-based cell tracking for the development and clinical use of hepatocyte transplantation methodologies. Further, these results generally highlight the importance of functional cell assays in the evaluation of contrast agent biocompatibility. PMID:25856627

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

    NASA Astrophysics Data System (ADS)

    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.

  7. Label-Retaining Cells in the Adult Murine Salivary Glands Possess Characteristics of Adult Progenitor Cells

    PubMed Central

    Chibly, Alejandro M.; Querin, Lauren; Harris, Zoey; Limesand, Kirsten H.

    2014-01-01

    Radiotherapy is the primary treatment for patients with head and neck cancer, which account for roughly 500,000 annual cases worldwide. Dysfunction of the salivary glands and associated conditions like xerostomia and dysphagia are often developed by these patients, greatly diminishing their life quality. Current preventative and palliative care fail to deliver an improvement in the quality of life, thus accentuating the need for regenerative therapies. In this study, a model of label retaining cells (LRCs) in murine salivary glands was developed, in which LRCs demonstrated proliferative potential and possessed markers of putative salivary progenitors. Mice were labeled with 5-Ethynyl-2′-deoxyuridine (EdU) at postnatal day 10 and chased for 8 weeks. Tissue sections from salivary glands obtained at the end of chase demonstrated co-localization between LRCs and the salivary progenitor markers keratin 5 and keratin 14, as well as kit mRNA, indicating that LRCs encompass a heterogeneous population of salivary progenitors. Proliferative potential of LRCs was demonstrated by a sphere assay, in which LRCs were found in primary and secondary spheres and they co-localized with the proliferation marker Ki67 throughout sphere formation. Surprisingly, LRCs were shown to be radio-resistant and evade apoptosis following radiation treatment. The clinical significance of these findings lie in the potential of this model to study the mechanisms that prevent salivary progenitors from maintaining homeostasis upon exposure to radiation, which will in turn facilitate the development of regenerative therapies for salivary gland dysfunction. PMID:25238060

  8. Label-retaining cells in the adult murine salivary glands possess characteristics of adult progenitor cells.

    PubMed

    Chibly, Alejandro M; Querin, Lauren; Harris, Zoey; Limesand, Kirsten H

    2014-01-01

    Radiotherapy is the primary treatment for patients with head and neck cancer, which account for roughly 500,000 annual cases worldwide. Dysfunction of the salivary glands and associated conditions like xerostomia and dysphagia are often developed by these patients, greatly diminishing their life quality. Current preventative and palliative care fail to deliver an improvement in the quality of life, thus accentuating the need for regenerative therapies. In this study, a model of label retaining cells (LRCs) in murine salivary glands was developed, in which LRCs demonstrated proliferative potential and possessed markers of putative salivary progenitors. Mice were labeled with 5-Ethynyl-2'-deoxyuridine (EdU) at postnatal day 10 and chased for 8 weeks. Tissue sections from salivary glands obtained at the end of chase demonstrated co-localization between LRCs and the salivary progenitor markers keratin 5 and keratin 14, as well as kit mRNA, indicating that LRCs encompass a heterogeneous population of salivary progenitors. Proliferative potential of LRCs was demonstrated by a sphere assay, in which LRCs were found in primary and secondary spheres and they co-localized with the proliferation marker Ki67 throughout sphere formation. Surprisingly, LRCs were shown to be radio-resistant and evade apoptosis following radiation treatment. The clinical significance of these findings lie in the potential of this model to study the mechanisms that prevent salivary progenitors from maintaining homeostasis upon exposure to radiation, which will in turn facilitate the development of regenerative therapies for salivary gland dysfunction.

  9. Label-Free and Continuous-Flow Ferrohydrodynamic Separation of HeLa Cells and Blood Cells in Biocompatible Ferrofluids

    PubMed Central

    Zhao, Wujun; Zhu, Taotao; Cheng, Rui; Liu, Yufei; He, Jian; Qiu, Hong; Wang, Lianchun; Nagy, Tamas; Querec, Troy D.; Unger, Elizabeth R.

    2016-01-01

    In this study, a label-free, low-cost, and fast ferrohydrodynamic cell separation scheme is demonstrated using HeLa cells (an epithelial cell line) and red blood cells. The separation is based on cell size difference, and conducted in a custom-made biocompatible ferrofluid that retains the viability of cells during and after the assay for downstream analysis. The scheme offers moderate-throughput (≈106 cells h−1 for a single channel device) and extremely high recovery rate (>99%) without the use of any label. It is envisioned that this separation scheme will have clinical applications in settings where rapid cell enrichment and removal of contaminating blood will improve efficiency of screening and diagnosis such as cervical cancer screening based on mixed populations in exfoliated samples. PMID:27478429

  10. Tumor cell differentiation by label-free microscopy

    NASA Astrophysics Data System (ADS)

    Schneckenburger, Herbert; Weber, Petra; Wagner, Michael

    2013-05-01

    Autofluorescence and Raman measurements of U251-MG glioblastoma cells prior and subsequent to activation of tumor suppressor genes are compared. While phase contrast images and fluorescence intensity patterns of the tumor (control) cells and the less malignant cells are similar, differences can be deduced from fluorescence spectra and nanosecond decay times. In particular, upon excitation around 375nm, the fluorescence ratio of the protein bound and the free coenzyme NADH depends on the state of malignancy and reflects different cytoplasmic (including lysosomal) and mitochondrial contributions. Slight differences are also observed in the Raman spectra of these cell lines, mainly originating from small granules (lysosomes) surrounding the cell nucleus. While larger numbers of fluorescence and Raman spectra are evaluated by multivariate statistical methods, additional information is obtained from spectral images and fluorescence lifetime images (FLIM).

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

    PubMed

    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.

  12. DNA cell-cycle analysis of cervical cancer by flow cytometry using simultaneous cytokeratin labelling for identification of tumour cells.

    PubMed

    Kimmig, R; Kapsner, T; Spelsberg, H; Untch, M; Hepp, H

    1995-01-01

    DNA ploidy and cell-cycle distribution were determined by flow cytometry in fresh tumour tissue of 53 cervical carcinomas. Epithelial cells were labelled by a fluorescein-isothiocyanate-conjugated cytokeratin antibody (CK6, CK18) to study the influence of contaminating stromal and inflammatory cells on results of cell-cycle analysis of tumour cells. Without identification of cytokeratin-positive cells 30/53 (57%) tumours were found to be DNA-aneuploid compared to 43/53 (81%) after gating for cytokeratin. Only 7 of 15 DNA-multiploid tumours could be detected without cytokeratin staining. In addition, cytokeratin-negative cells, which are found in all tumours, can be used as an internal standard for the calculation of ploidy and for quality control (coefficient of variation, linearity) of each individual sample. Cell-cycle analysis revealed significantly higher S-phase and G2M-phase fractions in cytokeratin-gated compared to ungated samples (13.1% versus 10.0% and 8.0% versus 5.4%; P < 0.001). This difference was more pronounced in DNA-diploid than DNA-aneuploid tumours. In conclusion, about 30% of DNA-aneuploid tumours could only be detected after cytokeratin labelling of epithelial cells. Owing to the identification of cytokeratin-positive cells the influence of non-tumoural cell elements on cell-cycle analysis was reduced markedly. Therefore, in cervical cancer, cytokeratin labelling can optimize both the determination of DNA ploidy and cell-cycle analysis.

  13. [Comparison of sorting of fluorescently and magnetically labelled dental pulp stem cells].

    PubMed

    Kerényi, Farkas; Tarapcsák, Szabolcs; Hrubi, Edit; Baráthne, Szabó Ágnes; Hegedüs, Viktória; Balogh, Sára; Bágyi, Kinga; Varga, Gábor; Hegedüs, Csaba

    2016-03-01

    Stem cells are present in many tissues, such as dental pulp. Stem cells can be easily isolated from dental pulp because third molars are often removed from patients. Stem cells could be separated from the tissue derived heterogeneous cell population. There are two main methods to separate a cell type from the other ones: the fluorescence activated cell sorting (FACS) and the magnetic activated cell sorting (MACS). The aim of this study was to compare these methods' effect on cell surviving and population growth after sorting on dental pulp cells. The anti-STRO-1 antibody was used as primary antibody to specifically label stem cells. Two secondary antibodies were used: magnetic or fluorescent labelled. We sorted the cells by MACS or by FACS or by combination of both (MACS-FACS). Our results show that the effectivity of MACS and FACS sorting are comparable while of MACS-FACS was significantly higher (MACS 79.53 ± 5.78%, FACS 88.27 ± 3.70%, MACS-FACS 98.43 ± 0.67%). The cell surviving and the post-sorting population growth, on the contrary, are very different. The cell population is growing on first week after MACS but after FACS did not. Moreover, after MACS-FACS, on first week the cell number of population decreased. Taken together, our results suggest to use MACS instead of FACS, at least in case of sorting dental pulp stem cells with anti-STRO-1 antibody. PMID:27188159

  14. Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles.

    PubMed

    Tefft, Brandon J; Uthamaraj, Susheil; Harburn, J Jonathan; Klabusay, Martin; Dragomir-Daescu, Dan; Sandhu, Gurpreet S

    2015-10-19

    Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

  15. Tumor cell differentiation by label-free fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Weber, Petra; Wagner, Michael; Kioschis, Petra; Kessler, Waltraud; Schneckenburger, Herbert

    2012-10-01

    Autofluorescence spectra, images, and decay kinetics of U251-MG glioblastoma cells prior and subsequent to activation of tumor suppressor genes are compared. While phase contrast images and fluorescence intensity patterns of tumor (control) cells and less malignant cells are similar, differences can be deduced from autofluorescence spectra and decay kinetics. In particular, upon near UV excitation, the fluorescence ratio of the free and protein-bound coenzyme nicotinamid adenine dinucleotide depends on the state of malignancy and reflects different cytoplasmic (including lysosomal) and mitochondrial contributions. While larger numbers of fluorescence spectra are evaluated by principal component analysis, a multivariate data analysis method, additional information on cell metabolism is obtained from spectral imaging and fluorescence lifetime imaging microscopy.

  16. Polar dibenzocyclooctynes for selective labeling of extracellular glycoconjugates of living cells.

    PubMed

    Friscourt, Frédéric; Ledin, Petr A; Mbua, Ngalle Eric; Flanagan-Steet, Heather R; Wolfert, Margreet A; Steet, Richard; Boons, Geert-Jan

    2012-03-21

    Although strain-promoted alkyne-azide cycloadditions (SPAAC) have found wide utility in biological and material sciences, the low polarity and limited water solubility of commonly used cyclooctynes represent a serious shortcoming. To address this problem, an efficient synthetic route has been developed for highly polar sulfated dibenzocyclooctynylamides (S-DIBO) by a Friedel-Crafts alkylation of 1,2-bis(3-methoxyphenyl)ethylamides with trichlorocyclopropenium cation followed by a controlled hydrolysis of the resulting dichlorocyclopropenes to give bis(3-methoxyphenyl)cyclooctacyclopropenones, which were subjected to methoxy group removal of the phenols, O-sulfation, and photochemical unmasking of the cyclopropenone moiety. Accurate rate measurements of the reaction of benzyl azide with various dibenzylcyclooctyne derivatives demonstrated that aromatic substitution and the presence of the amide function had only a marginal impact on the rate constants. Biotinylated S-DIBO 8 was successfully used for labeling azido-containing glycoconjugates of living cells. Furthermore, it was found that the substitution pattern of the dibenzylcyclooctynes influences subcellular location, and in particular it has been shown that DIBO derivative 4 can enter cells, thereby labeling intra- and extracellular azido-modified glycoconjugates, whereas S-DIBO 8 cannot pass the cell membrane and therefore is ideally suited for selective labeling of cell surface molecules. The ability to selectively label cell surface molecules will yield unique opportunities for glycomic analysis and the study of glycoprotein trafficking.

  17. Rapid Spectrophotometric Technique for Quantifying Iron in Cells Labeled with Superparamagnetic Iron Oxide Nanoparticles: Potential Translation to the Clinic

    PubMed Central

    Dadashzadeh, Esmaeel R.; Hobson, Matthew; Bryant, L. Henry; Dean, Dana D.; Frank, Joseph A.

    2012-01-01

    Labeling cells with superparamagnetic iron oxide (SPIO) nanoparticles provides the ability to track cells by Magnetic Resonance Imaging. Quantifying intracellular iron concentration in SPIO labeled cells would allow for the comparison of agents and techniques used to magnetically label cells. Here we describe a rapid spectrophotometric technique (ST) to quantify iron content of SPIO labeled cells, circumventing the previous requirement of an overnight acid digestion. Following lysis with 10% SDS of magnetically labeled cells, quantification of SPIO doped or labeled cells was performed using commonly available spectrophotometric instrument(s) by comparing absorptions at 370 and 750 nm with correction for turbidity of cellular products to determine iron content of each sample. Standard curves demonstrated high linear correlation (R2 = 0.998) between absorbance spectra of iron oxide nanoparticles and concentration in known SPIO doped cells. Comparisons of the ST to ICP-MS or NMR relaxometric (R2) determinations of intracellular iron contents in SPIO containing samples resulted in significant linear correlation between the techniques (R2 vs. ST, R2>0.992, p<0.0001, ST vs. ICP-MS, R2>0.995, p<0.0001) with the limit of detection of ST for iron = 0.66μg/ml. We have developed a rapid straightforward protocol that does not require overnight acid digestion for quantifying iron oxide content in magnetically labeled cells using readily available analytic instrumentation that should greatly expedite advances in comparing SPIO agents and protocols for labeling cells. PMID:23109392

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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.

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

    PubMed

    Gao, Feng; Gao, Tang; Zhou, Kechao; Zeng, Wenbin

    2016-01-01

    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. PMID:27589715

  1. Affordable uniform isotope labeling with (2)H, (13)C and (15)N in insect cells.

    PubMed

    Sitarska, Agnieszka; Skora, Lukasz; Klopp, Julia; Roest, Susan; Fernández, César; Shrestha, Binesh; Gossert, Alvar D

    2015-06-01

    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 (15)N and (13)C with yields comparable to expression in full media. For (2)H,(15)N and (2)H,(13)C,(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. PMID:25929326

  2. Magnetic resonance imaging of ferumoxide-labeled mesenchymal stem cells in cartilage defects: in vitro and in vivo investigations.

    PubMed

    Henning, Tobias D; Gawande, Rakhee; Khurana, Aman; Tavri, Sidhartha; Mandrussow, Lydia; Golovko, Daniel; Horvai, Andrew; Sennino, Barbara; McDonald, Donald; Meier, Reinhard; Wendland, Michael; Derugin, Nikita; Link, Thomas M; Daldrup-Link, Heike E

    2012-06-01

    The purpose of this study was to (1) compare three different techniques for ferumoxide labeling of mesenchymal stem cells (MSCs), (2) evaluate if ferumoxide labeling allows in vivo tracking of matrix-associated stem cell implants (MASIs) in an animal model, and (3) compare the magnetic resonance imaging (MRI) characteristics of ferumoxide-labeled viable and apoptotic MSCs. MSCs labeled with ferumoxide by simple incubation, protamine transfection, or Lipofectin transfection were evaluated with MRI and histopathology. Ferumoxide-labeled and unlabeled viable and apoptotic MSCs in osteochondral defects of rat knee joints were evaluated over 12 weeks with MRI. Signal to noise ratios (SNRs) of viable and apoptotic labeled MASIs were tested for significant differences using t-tests. A simple incubation labeling protocol demonstrated the best compromise between significant magnetic resonance signal effects and preserved cell viability and potential for immediate clinical translation. Labeled viable and apoptotic MASIs did not show significant differences in SNR. Labeled viable but not apoptotic MSCs demonstrated an increasing area of T2 signal loss over time, which correlated to stem cell proliferation at the transplantation site. Histopathology confirmed successful engraftment of viable MSCs. The engraftment of iron oxide-labeled MASIs by simple incubation can be monitored over several weeks with MRI. Viable and apoptotic MASIs can be distinguished via imaging signs of cell proliferation at the transplantation site. PMID:22554484

  3. Dye-doped organosilicate nanoparticles as cell-preserving labels for photoacoustic signal generation.

    PubMed

    Ramirez-Perez, Francisco I; Gutiérrez-Juárez Gerardo; Bok, Sangho; Gangopadhyay, Keshab; Gangopadhyay, Shubhra; Baker, Gary A; Polo-Parada, Luis

    2014-11-01

    Nanoparticle-assisted ultrasound generation by pulsed laser or photoacoustic (PA) techniques has been employed in the study of several tissues both in vivo and in vitro. Among the many applications of this technology, the detection of few cells in vitro is of particular interest. However, the toxicity induced by laser irradiation used for PA signal generation, whether in the absence or the presence of PA enhancers, within single isolated cells has not yet been investigated in detail. Herein, we report our studies of the cellular health of two different nanoparticle-labeled cell lines one hour after being subjected to a single laser pulse in vitro. We selected for this study an Hs936 skin epithelial melanoma cell line, which can be naturally detected photoacoustically, as well as a T47D human mammary breast gland epithelial cell line which has proven difficult to detect photoacoustically due to the absence of natural melanin. We have evaluated the amplitude of the PA signal derived from these two cell types, unlabeled and labeled with nanoparticles of two types (gold nanoparticles, AuNPs, or rhodamine 6G-doped organosilicate nanoparticles, R6G-NPOs), and assessed their health one hour subsequent to laser treatment. The current work corroborates previous findings that, for unlabeled cells, Hs936 produces a detectable PA signal whereas the T47D line does not. Cells labeled with AuNPs or R6G-NPOs produced a detectable PA signal of similar amplitude for the two cell lines. A significant number of Hs936 cells (both unlabeled cells and those labeled with AuNPs) exhibited cell nuclei alterations, as revealed by DAPI staining conducted an hour after photo treatment. Remarkably, the T47D cells suffered damage only when labeled with AuNPs. A significant finding, the R6G-NPOs proved capable of non-destructive PA signal generation in both cell types. Our findings advocate a transformational path forward for the use of dye-doped silicate nanoparticles in cell-compatible PA

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

    SciTech Connect

    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.

  5. Viability and MR detectability of iron labeled mesenchymal stem cells used for endoscopic injection into the porcine urethral sphincter.

    PubMed

    Will, Susanne; Martirosian, Petros; Eibofner, Frank; Schick, Fritz; Bantleon, Rüdiger; Vaegler, Martin; Grözinger, Gerd; Claussen, Claus D; Kramer, Ulrich; Schmehl, Jörg

    2015-08-01

    Direct stem cell therapies for functionally impaired tissue require a sufficient number of cells in the target region and a method for verifying the fate of the cells in the subsequent time course. In vivo MRI of iron labeled mesenchymal stem cells has been suggested to comply with these requirements. The study was conducted to evaluate proliferation, migration, differentiation and adhesion effects as well as the obtained iron load of an iron labeling strategy for mesenchymal stem cells. After injection into the porcine urethral sphincter, the labeled cells were monitored for up to six months using MRI. Mesenchymal stem cells were labeled with ferucarbotran (60/100/200 µg/mL) and ferumoxide (200 µg/mL) for the analysis of migration and viability. Phantom MR measurements were made to evaluate effects of iron labeling. For short and long term studies, the iron labeled cells were injected into the porcine urethral sphincter and monitored by MRI. High resolution anatomical images of the porcine urethral sphincter were applied for detection of the iron particles with a turbo-spin-echo sequence and a gradient-echo sequence with multiple TE values. The MR images were then compared with histological staining. The analysis of cell function after iron labeling showed no effects on proliferation or differentiation of the cells. Although the adherence increases with higher iron dose, the ability to migrate decreases as a presumed effect of iron labeling. The iron labeled mesenchymal stem cells were detectable in vivo in MRI and histological staining even six months after injection. Labeling with iron particles and subsequent evaluation with highly resolved three dimensional data acquisition allows sensitive tracking of cells injected into the porcine urethral sphincter for several months without substantial biological effects on mesenchymal stem cells.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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.

  8. Activity-dependent fluorescent labeling of bacterial cells expressing the TOL pathway

    SciTech Connect

    William K. Keener; Mary E. Watwood

    2005-01-01

    3-Ethynylbenzoate functions as an activity-dependent, fluorogenic and chromogenic probe for Pseudomonas putida mt-2, which is known to degrade toluene via conversion to benzoate, followed by meta ring fission of the intermediate, catechol. This direct physiological analysis allows the fluorescent labeling of cells whose toluene-degrading enzymes have been induced by an aromatic substrate.

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

    SciTech Connect

    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.

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

    PubMed

    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.

  11. Labeling of neuronal differentiation and neuron cells with biocompatible fluorescent nanodiamonds

    NASA Astrophysics Data System (ADS)

    Hsu, Tzu-Chia; Liu, Kuang-Kai; Chang, Huan-Cheng; Hwang, Eric; Chao, Jui-I.

    2014-05-01

    Nanodiamond is a promising carbon nanomaterial developed for biomedical applications. Here, we show fluorescent nanodiamond (FND) with the biocompatible properties that can be used for the labeling and tracking of neuronal differentiation and neuron cells derived from embryonal carcinoma stem (ECS) cells. The fluorescence intensities of FNDs were increased by treatment with FNDs in both the mouse P19 and human NT2/D1 ECS cells. FNDs were taken into ECS cells; however, FNDs did not alter the cellular morphology and growth ability. Moreover, FNDs did not change the protein expression of stem cell marker SSEA-1 of ECS cells. The neuronal differentiation of ECS cells could be induced by retinoic acid (RA). Interestingly, FNDs did not affect on the morphological alteration, cytotoxicity and apoptosis during the neuronal differentiation. Besides, FNDs did not alter the cell viability and the expression of neuron-specific marker β-III-tubulin in these differentiated neuron cells. The existence of FNDs in the neuron cells can be identified by confocal microscopy and flow cytometry. Together, FND is a biocompatible and readily detectable nanomaterial for the labeling and tracking of neuronal differentiation process and neuron cells from stem cells.

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

    PubMed

    Ramos-Gómez, Milagros; Martínez-Serrano, Alberto

    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 after in vivo transplantation. Magnetic resonance imaging provides an excellent non-invasive method to study the fate of transplanted cells in 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, proliferation, 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 engulfing dead labeled cells or iron nanoparticles dispersed freely in the brain parenchyma over time.

  13. Reconstruction of damaged corneal epithelium using Venus-labeled limbal epithelial stem cells and tracking of surviving donor cells.

    PubMed

    Yin, Ji-Qing; Liu, Wen-Qiang; Liu, Chao; Zhang, Yi-Hua; Hua, Jin-Lian; Liu, Wei-Shuai; Dou, Zhong-Ying; Lei, An-Min

    2013-10-01

    Limbal epithelial stem cells are responsible for the self-renewal and replenishment of the corneal epithelium. Although it is possible to repair the ocular surface using limbal stem cell transplantation, the mechanisms behind this therapy are unclear. To investigate the distribution of surviving donor cells in a reconstructed corneal epithelium, we screened a Venus-labeled limbal stem cell strain in goats. Cells were cultivated on denuded human amniotic membrane for 21 days to produce Venus-labeled corneal epithelial sheets. The Venus-labeled corneal epithelial sheets were transplanted to goat models of limbal stem cell deficiency. At 3 months post-surgery, the damaged corneal epithelia were obviously improved in the transplanted group compared with the non-transplanted control, with the donor cells still residing in the reconstructed ocular surface epithelium. Using Venus as a marker, our results indicated that the location and survival of donor cells varied, depending on the corneal epithelial region. Additionally, immunofluorescent staining of the reconstructed corneal epithelium demonstrated that many P63(+) cells were unevenly distributed among basal and suprabasal epithelial layers. Our study provides a new model, and reveals some of the mechanisms involved in corneal epithelial cell regeneration research.

  14. Learning from the Jersey Turnpike:Cell Lysis, Labeling and Washing with Microfluidic Metamaterials

    NASA Astrophysics Data System (ADS)

    Loutherback, Kevin; Morton, Keith; Inglis, David; Tsui, Opheli; Sturm, James; Chou, Stephen; Austin, Robert

    2008-03-01

    Directing objects across functional streamlines at low Reynolds number is difficult but important since this motion can be used to label, lyse, and analyze complex biological objects on-chip without cross-contamination. Here we use an asymmeteric post array to move cells across coflowing reagents and show on-chip, immunofluorescent labeling of platelets with washing and E.Coli cell lysis with simultaneous separation of bacterial chromosome from the cell contents. Furthermore, we develop the concept of a microfluidic metamaterial by using the basic asymmetric post array as a building block for complex particle handling modes. These modular array elements could be of great use for developing robust techniques for on-chip, continuous flow manipulation and analysis of cells, large bio-particles, and functional beads.

  15. Learning from the Jersey Turnpike: Cell Lysis, Labeling and Washing with Microfluidic Metamaterials

    NASA Astrophysics Data System (ADS)

    Austin, Robert

    2008-03-01

    Directing objects across functional streamlines at low Reynolds number is difficult but important since this motion can be used to label, lyse, and analyze complex biological objects on-chip without cross-contamination. Here we use an asymmeteric post array to move cells across coflowing reagents and show on-chip, immunofluorescent labeling of platelets with washing and E.Coli cell lysis with simultaneous separation of bacterial chromosome from the cell contents. Furthermore, we develop the concept of a microfluidic metamaterial by using the basic asymmetric post array as a building block for complex particle handling modes. These modular array elements could be of great use for developing robust techniques for on-chip, continuous flow manipulation and analysis of cells, large bio-particles, and functional beads.

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

    PubMed

    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

  17. Multi-luminescent hybrid gadolinium oxide nanoparticles as potential cell labeling.

    PubMed

    Fizet, J; Rivière, C; Bridot, J L; Charvet, N; Louis, C; Billotey, C; Raccurt, M; Morel, G; Roux, S; Perriat, P; Tillement, O

    2009-10-01

    This manuscript analyses the use of newly developed hybrid gadolinium oxide nanoparticles as cell-labeling tracers. The nanoparticles are core-shell particles composed of a core of gadolinium oxide of [2-4] nm and a protecting shell of polysiloxane [1-3 nm] where different organic dyes (fluoresceine isothiocyanate (FITC) or rhodamine B isothiocyanate (RBITC)) are embedded. They are functionalized with poly(ethylene glycol)bis(carboxymethyl) to ensure their colloidal stability in biological buffers. These particles are potential multi-labeling tracers (magnetic and optical). In this paper, we show by optical imaging that they can be efficiently internalized in cells without cell alteration. The in-vitro uptake of the nanoparticles was followed in two cell lines (human fibroblasts and a human adenocarnima cell lines MCF7 cells). Nanoparticles distribution within cells was analysed by confocal analysis, and gadolinium concentration within cells was quantified by mass spectrometry (ICP-MS analysis). Nanoparticles uptake is found to be fast and efficient for both cell lines, with fluorescent labeling visible after 10 min of incubation whatever the nature of the fluorophore. The fluorescent intensity is mainly found as concentrated dots in the perinuclear region of the cells and decreases with the number of days in culture, but is still easily detectable after 3 days in culture. No significant effect on cell growth was detected. Finally, we show in this study the protective effect of the polysiloxane layer: encapsulation of RBITC within the polysiloxane shell, leads to a better photostability of this low cost dye than Cy3 and even reach a level comparable to Alexa 595. With their high photostability and long-lasting contrast properties, these hybrid luminescent nanoparticles appears thus as a versatile solution to assess multiple cell fate both in in-vitro cell model as well as in-vivo.

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

    PubMed

    Yukawa, Hiroshi; Nakagawa, Shingo; Yoshizumi, Yasuma; Watanabe, Masaki; Saito, Hiroaki; Miyamoto, Yoshitaka; Noguchi, Hirofumi; Oishi, Koichi; Ono, Kenji; Sawada, Makoto; Kato, Ichiro; Onoshima, Daisuke; Obayashi, Momoko; Hayashi, Yumi; Kaji, Noritada; Ishikawa, Tetsuya; Hayashi, Shuji; Baba, Yoshinobu

    2014-01-01

    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. PMID:25365191

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

    NASA Astrophysics Data System (ADS)

    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.

  20. Label-free hyperspectral microscopy for scatter imaging of biological processes in cells (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Hwang, Jeeseong C.; Ray, Aniruddha; Cheney, Philip P.; Chon, Bonghwan; Lee, Ji Youn; Briggman, Kimberly A.

    2016-03-01

    We will present unique applications of a label-free, hyperspectral scatter imaging technique in different microscopy platforms including conventional wide-field, dark-field, and confocal. In different platforms, we conducted label-free imaging of cells undergoing biological processes such as nanoparticle uptake, apoptosis, and metabolic flux change in response to the variation of the osmotic pressure. Hyperspectral image analyses resolved spectral endmembers corresponding to unique scattering and absorption characteristics as a result of such processes at the single particle, single organelle, and single cell level, delineating the details of nanomaterial-cell interactions in a 2D cell culture, cell apoptotic characteristics in a 3D culture, and volumetric changes of single cells under the variation of osmotic pressure. Our label-free scatter imaging has the potential for a broad range of biological and biomedical applications such as the development of scatter-based imaging contrast agents and the measurement of scatter parameters of subcellular organelles to identify the sub-micron scale origins of scattering signals in tissue scattering measurements.

  1. A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

    PubMed Central

    Raghu, Deepa; Christodoulides, Joseph A.; Delehanty, James B.; Byers, Jeff M.; Raphael, Marc P.

    2015-01-01

    Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required. In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging. A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells. PMID:26650542

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

    NASA Astrophysics Data System (ADS)

    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.

  3. SIRB, sans iron oxide rhodamine B, a novel cross-linked dextran nanoparticle, labels human neuroprogenitor and SH-SY5Y neuroblastoma cells and serves as a USPIO cell labeling control.

    PubMed

    Shen, Wei-Bin; Vaccaro, Dennis E; Fishman, Paul S; Groman, Ernest V; Yarowsky, Paul

    2016-05-01

    This is the first report of the synthesis of a new nanoparticle, sans iron oxide rhodamine B (SIRB), an example of a new class of nanoparticles. SIRB is designed to provide all of the cell labeling properties of the ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle Molday ION Rhodamine B (MIRB) without containing the iron oxide core. MIRB was developed to label cells and allow them to be tracked by MRI or to be manipulated by magnetic gradients. SIRB possesses a similar size, charge and cross-linked dextran coating as MIRB. Of great interest is understanding the biological and physiological changes in cells after they are labeled with a USPIO. Whether these effects are due to the iron oxide buried within the nanoparticle or to the surface coating surrounding the iron oxide core has not been considered previously. MIRB and SIRB represent an ideal pairing of nanoparticles to identify nanoparticle anatomy responsible for post-labeling cytotoxicity. Here we report the effects of SIRB labeling on the SH-SY5Y neuroblastoma cell line and primary human neuroprogenitor cells (hNPCs). These effects are contrasted with the effects of labeling SH-SY5Y cells and hNPCs with MIRB. We find that SIRB labeling, like MIRB labeling, (i) occurs without the use of transfection reagents, (ii) is packaged within lysosomes distributed within cell cytoplasm, (iii) is retained within cells with no loss of label after cell storage, and (iv) does not alter cellular viability or proliferation, and (v) SIRB labeled hNPCs differentiate normally into neurons or astrocytes. Copyright © 2016 John Wiley & Sons, Ltd.

  4. SIRB, sans iron oxide rhodamine B, a novel cross-linked dextran nanoparticle, labels human neuroprogenitor and SH-SY5Y neuroblastoma cells and serves as a USPIO cell labeling control.

    PubMed

    Shen, Wei-Bin; Vaccaro, Dennis E; Fishman, Paul S; Groman, Ernest V; Yarowsky, Paul

    2016-05-01

    This is the first report of the synthesis of a new nanoparticle, sans iron oxide rhodamine B (SIRB), an example of a new class of nanoparticles. SIRB is designed to provide all of the cell labeling properties of the ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle Molday ION Rhodamine B (MIRB) without containing the iron oxide core. MIRB was developed to label cells and allow them to be tracked by MRI or to be manipulated by magnetic gradients. SIRB possesses a similar size, charge and cross-linked dextran coating as MIRB. Of great interest is understanding the biological and physiological changes in cells after they are labeled with a USPIO. Whether these effects are due to the iron oxide buried within the nanoparticle or to the surface coating surrounding the iron oxide core has not been considered previously. MIRB and SIRB represent an ideal pairing of nanoparticles to identify nanoparticle anatomy responsible for post-labeling cytotoxicity. Here we report the effects of SIRB labeling on the SH-SY5Y neuroblastoma cell line and primary human neuroprogenitor cells (hNPCs). These effects are contrasted with the effects of labeling SH-SY5Y cells and hNPCs with MIRB. We find that SIRB labeling, like MIRB labeling, (i) occurs without the use of transfection reagents, (ii) is packaged within lysosomes distributed within cell cytoplasm, (iii) is retained within cells with no loss of label after cell storage, and (iv) does not alter cellular viability or proliferation, and (v) SIRB labeled hNPCs differentiate normally into neurons or astrocytes. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26809657

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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

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

    PubMed

    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.

  8. Chemically Activatable Alkyne Tag for Low pH-Enhanced Molecular Labeling on Living Cells.

    PubMed

    Yamaguchi, Satoshi; Ura, Manami; Izuta, Shin; Okamoto, Akimitsu

    2016-09-21

    Stimuli-responsive "activatable" reactive tags are applicable to selective labeling of biomolecules in a defined area or environment in living systems, yielding new insights into cellular processes through molecular imaging and fishing. Here, we developed a chemically activatable alkyne tag that can be incorporated into biological molecules and labeled with azide-tagged functional molecules through the alkyne-azide cycloaddition "click" reaction after chemical activation. Formation of the alkyne tag from the precursor moiety was confirmed to proceed in physiological aqueous media and was particularly enhanced under mildly acidic pH. The tag was successfully applied to low-pH sensitive labeling of a cholesterol analogue with azide-tagged biotin on living mammalian cells. Our results provided proof of principle that the present activatable alkyne tag can be used as a tool to selectively analyze molecules of interest in low-pH regions in living systems. PMID:27526276

  9. Comparison of alternative nucleophiles for Sortase A-mediated bioconjugation and application in neuronal cell labelling.

    PubMed

    Baer, Samuel; Nigro, Julie; Madej, Mariusz P; Nisbet, Rebecca M; Suryadinata, Randy; Coia, Gregory; Hong, Lisa P T; Adams, Timothy E; Williams, Charlotte C; Nuttall, Stewart D

    2014-05-01

    The Sortase A (SrtA) enzyme from Staphylococcus aureus catalyses covalent attachment of protein substrates to pentaglycine cross-bridges in the Gram positive bacterial cell wall. In vitro SrtA-mediated protein ligation is now an important protein engineering tool for conjugation of substrates containing the LPXTGX peptide recognition sequence to oligo-glycine nucleophiles. In order to explore the use of alternative nucleophiles in this system, five different rhodamine-labelled compounds, with N-terminal nucleophilic amino acids, triglycine, glycine, and lysine, or N-terminal non-amino acid nucleophiles ethylenediamine and cadaverine, were synthesized. These compounds were tested for their relative abilities to function as nucleophiles in SrtA-mediated bioconjugation reactions. N-Terminal triglycine, glycine and ethylenediamine were all efficient in labelling a range of LPETGG containing recombinant antibody and scaffold proteins and peptides, while reduced activity was observed for the other nucleophiles across the range of proteins and peptides studied. Expansion of the range of available nucleophiles which can be utilised in SrtA-mediated bioconjugation expands the range of potential applications for this technology. As a demonstration of the utility of this system, SrtA coupling was used to conjugate the triglycine rhodamine-labelled nucleophile to the C-terminus of an Im7 scaffold protein displaying Aβ, a neurologically important peptide implicated in Alzheimer's disease. Purified, labelled protein showed Aβ-specific targeting to mammalian neuronal cells. Demonstration of targeting neuronal cells with a chimeric protein illustrates the power of this system, and suggests that SrtA-mediated direct cell-surface labelling and visualisation is an achievable goal. PMID:24643508

  10. Heterogeneity of basal keratinocytes: nonrandom distribution of thymidine-labeled basal cells in confluent cultures is not a technical artifact

    SciTech Connect

    Milstone, L.M.; LaVigne, J.F.

    1985-06-01

    Basal surface autoradiography of (/sup 3/H)dThd-labeled, confluent, keratinocyte cultures reveals that proliferating cells have a nonrandom, patterned distribution. Unlabeled cells, likewise, appear nonrandomly in clusters. The authors show here that failure to detect DNA synthesis in some basal cells in culture is not an artifact caused either by physical separation of the labeled nuclei from the radiographic emulsion or by a diffusion barrier that would prevent (/sup 3/H)dThd from reaching basal cells.

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

    SciTech Connect

    Brysk, M.M.; Snider, J.M.

    1982-05-01

    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 /sup 125/I-lectins, and by the metabolic incorporation of L-(/sup 3/H)-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 /sup 125/I-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.

  12. Vital fluorescent labeling for confocal scanning microscopic study of living cell invasion

    NASA Astrophysics Data System (ADS)

    Wang, Allan Z.; Chen, Jian M.; Fisher, Gregory W.; Wang, Jane C.

    1997-07-01

    Invasion by cells with malignant or transformed phenotypes precedes destruction of adjacent tissue and fatal cell metastasis. State-of-the-art confocal laser scanning technology facilitates both in vitro and in vivo research into cell invasion and metastasis. In particular, studies performed with living cells yield more precise information than those with fixed cells, giving new insight into cell invasion and metastasis. We have tested a variety of vital florescent dyes and fluorogenic protease substrates in our studies of invasion of cartilage by transformed synoviocytes or osteosarcoma cells. The fluorescent dyes tested include Calcein acetoxy methyl-FITC (Calcein), Hoechst 33342 (Hoechst), CellTracker, DiI, DiO, DiD, and ethidium bromide (EB). The fluorogenic protease substrate used Meoxysuccinyl-Gly-Pro-Leu-Gly-Pro-AFC (MOS-GPLGP-AFC) for detection of collagenase activity. We found that Calcein-FITC labeling permitted the clearest direct observation of the penetration of transformed synoviocytes and osteosarcoma cells into cartilage. Even better results were obtained when chondrocyte nuclei were counter-stained with Hoechst 33342. During the invasion process, collagenase activity was observed around the synoviocyte in the cartilage matrix labeled with the fluorogenic collagenase substrate. We concluded that of the vital fluorescent dyes tested, a combined application of Calcein-FITC, Hoechst 23223, and MOS- GPLGP-AFC is most appropriate for the study of the cell invasion process.

  13. Current approaches for RNA labeling in vitro and in cells based on click reactions.

    PubMed

    Schulz, Daniela; Rentmeister, Andrea

    2014-11-01

    Over recent years, click reactions have become recognized as valuable and flexible tools to label biomacromolecules such as proteins, nucleic acids, and glycans. Some of the developed strategies can be performed not only in aqueous solution but also in the presence of cellular components, as well as on (or even in) living cells. These labeling strategies require the initial, specific modification of the target molecule with a small, reactive moiety. In the second step, a click reaction is used to covalently couple a reporter molecule to the biomolecule. Depending on the type of reporter, labeling by the click reaction can be used in many different applications, ranging from isolation to functional studies of biomacromolecules. In this minireview, we focus on labeling strategies for RNA that rely on the click reaction. We first highlight click reactions that have been used successfully to label modified RNA, and then describe different strategies to introduce the required reactive groups into target RNA. The benefits and potential limitations of the strategies are critically discussed with regard to possible future developments. PMID:25224574

  14. Genetically-Directed, Cell Type-Specific Sparse Labeling for the Analysis of Neuronal Morphology

    PubMed Central

    Rotolo, Thomas; Smallwood, Philip M.; Williams, John; Nathans, Jeremy

    2008-01-01

    Background In mammals, genetically-directed cell labeling technologies have not yet been applied to the morphologic analysis of neurons with very large and complex arbors, an application that requires extremely sparse labeling and that is only rendered practical by limiting the labeled population to one or a few predetermined neuronal subtypes. Methods and Findings In the present study we have addressed this application by using CreER technology to non-invasively label very small numbers of neurons so that their morphologies can be fully visualized. Four lines of IRES-CreER knock-in mice were constructed to permit labeling selectively in cholinergic or catecholaminergic neurons [choline acetyltransferase (ChAT)-IRES-CreER or tyrosine hydroxylase (TH)-IRES-CreER], predominantly in projection neurons [neurofilament light chain (NFL)-IRES-CreER], or broadly in neurons and some glia [vesicle-associated membrane protein2 (VAMP2)-IRES-CreER]. When crossed to the Z/AP reporter and exposed to 4-hydroxytamoxifen in the early postnatal period, the number of neurons expressing the human placental alkaline phosphatase reporter can be reproducibly lowered to fewer than 50 per brain. Sparse Cre-mediated recombination in ChAT-IRES-CreER;Z/AP mice shows the full axonal and dendritic arbors of individual forebrain cholinergic neurons, the first time that the complete morphologies of these very large neurons have been revealed in any species. Conclusions Sparse genetically-directed, cell type-specific neuronal labeling with IRES-creER lines should prove useful for studying a wide variety of questions in neuronal development and disease. PMID:19116659

  15. Label-free cell cycle analysis for high-throughput imaging flow cytometry

    PubMed Central

    Blasi, Thomas; Hennig, Holger; Summers, Huw D.; Theis, Fabian J.; Cerveira, Joana; Patterson, James O.; Davies, Derek; Filby, Andrew; Carpenter, Anne E.; Rees, Paul

    2016-01-01

    Imaging flow cytometry combines the high-throughput capabilities of conventional flow cytometry with single-cell imaging. Here we demonstrate label-free prediction of DNA content and quantification of the mitotic cell cycle phases by applying supervised machine learning to morphological features extracted from brightfield and the typically ignored darkfield images of cells from an imaging flow cytometer. This method facilitates non-destructive monitoring of cells avoiding potentially confounding effects of fluorescent stains while maximizing available fluorescence channels. The method is effective in cell cycle analysis for mammalian cells, both fixed and live, and accurately assesses the impact of a cell cycle mitotic phase blocking agent. As the same method is effective in predicting the DNA content of fission yeast, it is likely to have a broad application to other cell types. PMID:26739115

  16. Label-free detection of cell-contractile activity with lipid nanotubes.

    PubMed

    Sugihara, Kaori; Delai, Marco; Mahnna, Rami; Kusch, Justine; Poulikakos, Dimos; Vörös, János; Zambelli, Tomaso; Ferrari, Aldo

    2013-02-01

    Surface-bound self-assembled lipid nanotubes (LNTs) made of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were used to visualize the contractile activity of spreading cells. The interaction of cells with LNTs resulted in the nucleation of new nanotubes, directed toward the cell center, from existing ones. This process depended on cell generated forces and required acto-myosin mediated contractility. The dynamics of de novo generation of LNTs upon cell spreading was captured using optical microscopy on fluorescently labeled nanotubes and revealed characteristic fingerprints for different cell types such as fibroblasts, endothelial and melanoma cells. Additionally, the method was applied to detect the effect of a specific inhibitor on the generation of cellular forces. The mechanism of the LNT-cell interaction and the potential applications are discussed.

  17. High-resolution x-ray diffraction microscopy of specifically labeled yeast cells

    DOE PAGESBeta

    Nelson, Johanna; Huang, Xiaojing; Steinbrener, Jan; Shapiro, David; Kirz, Janos; Marchesini, Stephano; Neiman, Aaron M.; Turner, Joshua J.; Jacobsen, Chris

    2010-04-20

    X-ray diffraction microscopy complements other x-ray microscopy methods by being free of lens-imposed radiation dose and resolution limits, and it allows for high-resolution imaging of biological specimens too thick to be viewed by electron microscopy. We report here the highest resolution (11-13 nm) x-ray diffraction micrograph of biological specimens, and a demonstration of molecular-specific gold labeling at different depths within cells via through-focus propagation of the reconstructed wavefield. The lectin concanavalin A conjugated to colloidal gold particles was used to label the α-mannan sugar in the cell wall of the yeast Saccharomyces cerevisiae. Cells were plunge-frozen in liquid ethane andmore » freeze-dried, after which they were imaged whole using x-ray diffraction microscopy at 750 eV photon energy.« less

  18. High-resolution x-ray diffraction microscopy of specifically labeled yeast cells

    SciTech Connect

    Nelson, Johanna; Huang, Xiaojing; Steinbrener, Jan; Shapiro, David; Kirz, Janos; Marchesini, Stephano; Neiman, Aaron M.; Turner, Joshua J.; Jacobsen, Chris

    2010-04-20

    X-ray diffraction microscopy complements other x-ray microscopy methods by being free of lens-imposed radiation dose and resolution limits, and it allows for high-resolution imaging of biological specimens too thick to be viewed by electron microscopy. We report here the highest resolution (11-13 nm) x-ray diffraction micrograph of biological specimens, and a demonstration of molecular-specific gold labeling at different depths within cells via through-focus propagation of the reconstructed wavefield. The lectin concanavalin A conjugated to colloidal gold particles was used to label the α-mannan sugar in the cell wall of the yeast Saccharomyces cerevisiae. Cells were plunge-frozen in liquid ethane and freeze-dried, after which they were imaged whole using x-ray diffraction microscopy at 750 eV photon energy.

  19. Cell labeling and magnetic separation by means of immunoreagents based on polyacrolein microspheres.

    PubMed

    Rembaum, A; Yen, R C; Kempner, D H; Ugelstad, J

    1982-08-13

    Polyacrolein (PA) microspheres were synthesized by means of ionizing radiation and shown to contain aldehyde groups which form covalent bounds with amino compounds and proteins. PA microspheres made fluorescent after reaction with fluorescein-labeled antibodies were found to specifically label sensitized sheep red blood cells (SRBC). PA microspheres could also be grafted onto a variety of polymeric spheres of different sizes and composition by ionizing radiation. These hybrid spheres, i.e., preformed polymeric spheres with PA microspheres grafted on their surfaces could bind antibodies which retained specificity of reaction with cell surface receptors. Purification of sensitized SRBC from a mixture containing chicken red blood cells (CRBC) by means of hybrids magnetic spheres in a magnetic field was demonstrated. PMID:7130709

  20. Ultrasound and photoacoustic imaging to monitor mesenchymal stem cells labeled with gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Nam, Seung Yun; Ricles, Laura M.; Sokolov, Konstantin; Suggs, Laura J.; Emelianov, Stanislav Y.

    2011-03-01

    Mesenchymal stem cells (MSCs) are versatile in many tissue engineering applications and have the potential to be used for cellular therapies because they can differentiate into many cell types. Specifically, the use of MSCs for the treatment of ischemic disease is promising because MSCs can express characteristics of vascular cells. MSCs can promote vascular growth at the site of injury after delivery using a PEGylated fibrin gel. In order to quantitatively assess in vivo delivery and differentiation of MSCs, a non-invasive and high-resolution imaging technique is required. In this study, the combined ultrasound and photoacoustic imaging was demonstrated to monitor MSCs labeled with citrate-stabilized gold nanoparticles (Au NPs). It was observed that uptake of nanoparticles did not have a significant effect on cell viability and proliferation over a two-week period. Four different cell concentrations of either the non-labeled MSCs or the Au NP labeled MSCs were embedded in the tissue mimicking gelatin phantom. The ultrasound and photoacoustic signals were acquired and quantitatively analyzed to assess sensitivity and accuracy of the developed imaging approach. Furthermore, based on the results, the feasibility of in vivo ultrasound and photoacoustic imaging of MSCs was discussed.

  1. Proteinases release /sup 35/S-labeled macromolecules from cultured airway epithelial cells

    SciTech Connect

    Varsano, S.; Borson, D.B.; Gold, M.; Forsberg, S.; Basbaum, C.B.; Nadel, J.A.

    1986-03-05

    To determine whether proteinases release radiolabeled macromolecules from airway cells devoid of secretory granules, they studied canine cultured tracheal epithelial cells grown to confluency. At this time the cells are bound by tight junctions, maintain anion transport, have a well developed glycocalyx, but contain no secretory granules. They labeled the cells with /sup 35/SO/sub 4/ (50..mu..ci/ml/24h) then changed the medium every 20 min and measured nondialyzable /sup 35/S released into the medium. Two h later, the rate of spontaneous release of /sup 35/S-labeled-macromolecules was 5700 +/- 1600 CPM/20 min (mean +/- SD). At this time trypsin, thermolysin, pseudomonas elastase and alkaline proteinase, each released /sup 35/S-labeled-macromolecules, whereas aspergillus acid proteinase did not. In more detailed studies, trypsin released /sup 35/S in a concentration dependent fashion, with a threshold below 10 units/ml and a response to 1000 units/ml of 1092 +/- 173% (mean +/- SD; n=5 cultures) above pre-trypsin baseline. Sepharose CL4B chromatography of the radiolabeled materials released by trypsin showed a void volume fraction (MW greater than or equal to 10/sup 6/), and a second, included fraction (MW 2-3 x 10/sup 5/). These results indicate that cultured airway epithelial cells synthesize macromolecules and release them into the medium, and that proteinases increase the rate of macromolecule release markedly.

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

    PubMed

    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

  3. Correlative fluorescence and scanning transmission electron microscopy of quantum dot-labeled proteins on whole cells in liquid.

    PubMed

    Peckys, Diana B; Bandmann, Vera; de Jonge, Niels

    2014-01-01

    Correlative fluorescence microscopy combined with scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, STEM can be accomplished in two ways. The microchip with the labeled cells and one microchip with a spacer are assembled into a special microfluidic device and imaged with dedicated high-voltage STEM. Alternatively, thin edges of cells can be studied with environmental scanning electron microscopy with a STEM detector, by placing a microchip with cells in a cooled wet environment.

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

    PubMed

    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

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

    PubMed Central

    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

  6. Incorporation and turnover of biotin-labeled actin microinjected into fibroblastic cells: an immunoelectron microscopic study

    PubMed Central

    1989-01-01

    We investigated the mechanism of turnover of an actin microfilament system in fibroblastic cells on an electron microscopic level. A new derivative of actin was prepared by labeling muscle actin with biotin. Cultured fibroblastic cells were microinjected with biotinylated actin, and incorporated biotin-actin molecules were detected by immunoelectron microscopy using an anti-biotin antibody and a colloidal gold-labeled secondary antibody. We also analyzed the localization of injected biotin-actin molecules on a molecular level by freeze-drying techniques. Incorporation of biotin-actin was rapid in motile peripheral regions, such as lamellipodia and microspikes. At approximately 1 min after injection, biotin-actin molecules were mainly incorporated into the distal part of actin bundles in the microspikes. Heavily labeled actin filaments were also observed at the distal fringe of the densely packed actin networks in the lamellipodium. By 5 min after injection, most actin polymers in microspikes and lamellipodia were labeled uniformly. These findings suggest that actin subunits are added preferentially at the membrane-associated ends of preexisting actin filaments. At earlier times after injection, we often observed that the labeled segments were continuous with unlabeled segments, suggesting the incorporation of new subunits at the ends of preexisting filaments. Actin incorporation into stress fibers was a slower process. At 2-3 min after injection, microfilaments at the surface of stress fibers incorporated biotin-actin, but filaments in the core region of stress fibers did not. At 5-10 min after injection, increasing density of labeling along stress fibers toward their distal ends was observed. Stress fiber termini are generally associated with focal contacts. There was no rapid nucleation of actin filaments off the membrane of focal contacts and the pattern of actin incorporation at focal contacts was essentially identical to that into distal parts of stress fibers

  7. Bioorthogonal labeling cell-surface proteins expressed in pancreatic cancer cells to identify potential diagnostic/therapeutic biomarkers

    PubMed Central

    Haun, Randy S; Quick, Charles M; Siegel, Eric R; Raju, Ilangovan; Mackintosh, Samuel G; Tackett, Alan J

    2015-01-01

    To develop new diagnostic and therapeutic tools to specifically target pancreatic tumors, it is necessary to identify cell-surface proteins that may serve as potential tumor-specific targets. In this study we used an azido-labeled bioorthogonal chemical reporter to metabolically label N-linked glycoproteins on the surface of pancreatic cancer cell lines to identify potential targets that may be exploited for detection and/or treatment of pancreatic cancer. Labeled glycoproteins were tagged with biotin using click chemistry, purified by streptavidin-coupled magnetic beads, separated by gel electrophoresis, and identified by liquid chromatography-tandem mass spectrometry (MS). MS/MS analysis of peptides from 3 cell lines revealed 954 unique proteins enriched in the azido sugar samples relative to control sugar samples. A comparison of the proteins identified in each sample indicated 20% of these proteins were present in 2 cell lines (193 of 954) and 17 of the proteins were found in all 3 cell lines. Five of the 17 proteins identified in all 3 cell lines have not been previously reported to be expressed in pancreatic cancer; thus indicating that novel cell-surface proteins can be revealed through glycoprotein profiling. Western analysis of one of these glycoproteins, ecto-5′-nucleotidase (NT5E), revealed it is expressed in 8 out of 8 pancreatic cancer cell lines examined. Further, immunohistochemical analysis of human pancreatic tissues indicates NT5E is significantly overexpressed in pancreatic tumors compared to normal pancreas. Thus, we have demonstrated that metabolic labeling with bioorthogonal chemical reporters can be used to selectively enrich and identify novel cell-surface glycoproteins expressed in pancreatic ductal adenocarcinomas. PMID:26176765

  8. Production of stable isotope-labeled acyl-coenzyme A thioesters by yeast stable isotope labeling by essential nutrients in cell culture.

    PubMed

    Snyder, Nathaniel W; Tombline, Gregory; Worth, Andrew J; Parry, Robert C; Silvers, Jacob A; Gillespie, Kevin P; Basu, Sankha S; Millen, Jonathan; Goldfarb, David S; Blair, Ian A

    2015-04-01

    Acyl-coenzyme A (CoA) thioesters are key metabolites in numerous anabolic and catabolic pathways, including fatty acid biosynthesis and β-oxidation, the Krebs cycle, and cholesterol and isoprenoid biosynthesis. Stable isotope dilution-based methodology is the "gold standard" for quantitative analyses by mass spectrometry. However, chemical synthesis of families of stable isotope-labeled metabolites such as acyl-CoA thioesters is impractical. Previously, we biosynthetically generated a library of stable isotope internal standard analogs of acyl-CoA thioesters by exploiting the essential requirement in mammals and insects for pantothenic acid (vitamin B5) as a metabolic precursor for the CoA backbone. By replacing pantothenic acid in the cell medium with commercially available [(13)C3(15)N1]-pantothenic acid, mammalian cells exclusively incorporated [(13)C3(15)N1]-pantothenate into the biosynthesis of acyl-CoA and acyl-CoA thioesters. We have now developed a much more efficient method for generating stable isotope-labeled CoA and acyl-CoAs from [(13)C3(15)N1]-pantothenate using stable isotope labeling by essential nutrients in cell culture (SILEC) in Pan6-deficient yeast cells. Efficiency and consistency of labeling were also increased, likely due to the stringently defined and reproducible conditions used for yeast culture. The yeast SILEC method greatly enhances the ease of use and accessibility of labeled CoA thioesters and also provides proof of concept for generating other labeled metabolites in yeast mutants.

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

    PubMed

    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.

  10. Hydrodynamic and label-free sorting of circulating tumor cells from whole blood

    NASA Astrophysics Data System (ADS)

    Geislinger, Thomas M.; Stamp, Melanie E. M.; Wixforth, Achim; Franke, Thomas

    2015-11-01

    We demonstrate continuous, passive, and label-free sorting of different in vitro cancer cell lines (MV3, MCF7, and HEPG2) as model systems for circulating tumor cells (CTCs) from undiluted whole blood employing the non-inertial lift effect as driving force. This purely viscous, repulsive cell-wall interaction is sensitive to cell size and deformability differences and yields highly efficient cell separation and high enrichment factors. We show that the performance of the device is robust over a large range of blood cell concentrations and flow rates as well as for the different cell lines. The collected samples usually contain more than 90% of the initially injected CTCs and exhibit average enrichment factors of more than 20 for sorting from whole blood samples.

  11. In vivo capture and label-free detection of early metastatic cells

    PubMed Central

    Azarin, Samira M.; Yi, Ji; Gower, Robert M.; Aguado, Brian A.; Sullivan, Megan E.; Goodman, Ashley G.; Jiang, Eric J.; Rao, Shreyas S.; Ren, Yinying; Tucker, Susan L.; Backman, Vadim; Jeruss, Jacqueline S.; Shea, Lonnie D.

    2015-01-01

    Breast cancer is a leading cause of death for women, with mortality resulting from metastasis. Metastases are often detected once tumor cells affect the function of solid organs, with a high disease burden limiting effective treatment. Here we report a method for the early detection of metastasis using an implanted scaffold to recruit and capture metastatic cells in vivo, which achieves high cell densities and reduces the tumor burden within solid organs 10-fold. Recruitment is associated with infiltration of immune cells, which include Gr1hiCD11b+ cells. We identify metastatic cells in the scaffold through a label-free detection system using inverse-spectroscopic optical coherence tomography, which identifies changes to nanoscale tissue architecture associated with the presence of tumor cells. For patients at risk of recurrence, scaffold implantation following completion of primary therapy has the potential to identify metastatic disease at the earliest stage, enabling initiation of therapy while the disease burden is low. PMID:26348915

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

    PubMed

    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.

  13. Analysis of Cell-Surface Receptor Dynamics through Covalent Labeling by Catalyst-Tethered Antibody.

    PubMed

    Hayashi, Takahiro; Yasueda, Yuki; Tamura, Tomonori; Takaoka, Yousuke; Hamachi, Itaru

    2015-04-29

    A general technique for introducing biophysical probes into selected receptors in their native environment is valuable for the study of their structure, dynamics, function, and molecular interactions. A number of such techniques rely on genetic engineering, which is not applicable for the study of endogenous proteins, and such approaches often suffer from artifacts due to the overexpression and bulky size of the probes/protein tags used. Here we designed novel catalyst-antibody conjugates capable of introducing small chemical probes into receptor proteins such as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) in a selective manner on the surface of living cells. Because of the selectivity and efficiency of this labeling technique, we were able to monitor the cellular dynamics and lifetime of HER2 endogenously expressed on cancer cells. More significantly, the current labeling technique comprises a stable covalent bond, which combined with a peptide mass fingerprinting analysis allowed epitope mapping of antibodies on living cells and identification of potential binding sites of anti-EGFR affibody. Although as yet unreported in the literature, the binding sites predicted by our labeling method were consistently supported by the subsequent mutation and binding assay experiments. In addition, this covalent labeling method provided experimental evidence that HER2 exhibits a more dynamic structure than expected on the basis of crystallographic analysis alone. Our novel catalyst-antibody conjugates are expected to provide a general tool for investigating the protein trafficking, fluctuation, and molecular interactions of an important class of cell-surface receptors on live cell surfaces. PMID:25853648

  14. Analysis of Cell-Surface Receptor Dynamics through Covalent Labeling by Catalyst-Tethered Antibody.

    PubMed

    Hayashi, Takahiro; Yasueda, Yuki; Tamura, Tomonori; Takaoka, Yousuke; Hamachi, Itaru

    2015-04-29

    A general technique for introducing biophysical probes into selected receptors in their native environment is valuable for the study of their structure, dynamics, function, and molecular interactions. A number of such techniques rely on genetic engineering, which is not applicable for the study of endogenous proteins, and such approaches often suffer from artifacts due to the overexpression and bulky size of the probes/protein tags used. Here we designed novel catalyst-antibody conjugates capable of introducing small chemical probes into receptor proteins such as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) in a selective manner on the surface of living cells. Because of the selectivity and efficiency of this labeling technique, we were able to monitor the cellular dynamics and lifetime of HER2 endogenously expressed on cancer cells. More significantly, the current labeling technique comprises a stable covalent bond, which combined with a peptide mass fingerprinting analysis allowed epitope mapping of antibodies on living cells and identification of potential binding sites of anti-EGFR affibody. Although as yet unreported in the literature, the binding sites predicted by our labeling method were consistently supported by the subsequent mutation and binding assay experiments. In addition, this covalent labeling method provided experimental evidence that HER2 exhibits a more dynamic structure than expected on the basis of crystallographic analysis alone. Our novel catalyst-antibody conjugates are expected to provide a general tool for investigating the protein trafficking, fluctuation, and molecular interactions of an important class of cell-surface receptors on live cell surfaces.

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

    PubMed

    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

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

    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.

  17. Label free cell tracking in 3D tissue engineering constructs with high resolution imaging

    NASA Astrophysics Data System (ADS)

    Smith, W. A.; Lam, K.-P.; Dempsey, K. P.; Mazzocchi-Jones, D.; Richardson, J. B.; Yang, Y.

    2014-02-01

    Within the field of tissue engineering there is an emphasis on studying 3-D live tissue structures. Consequently, to investigate and identify cellular activities and phenotypes in a 3-D environment for all in vitro experiments, including shape, migration/proliferation and axon projection, it is necessary to adopt an optical imaging system that enables monitoring 3-D cellular activities and morphology through the thickness of the construct for an extended culture period without cell labeling. This paper describes a new 3-D tracking algorithm developed for Cell-IQ®, an automated cell imaging platform, which has been equipped with an environmental chamber optimized to enable capturing time-lapse sequences of live cell images over a long-term period without cell labeling. As an integral part of the algorithm, a novel auto-focusing procedure was developed for phase contrast microscopy equipped with 20x and 40x objectives, to provide a more accurate estimation of cell growth/trajectories by allowing 3-D voxels to be computed at high spatiotemporal resolution and cell density. A pilot study was carried out in a phantom system consisting of horizontally aligned nanofiber layers (with precise spacing between them), to mimic features well exemplified in cellular activities of neuronal growth in a 3-D environment. This was followed by detailed investigations concerning axonal projections and dendritic circuitry formation in a 3-D tissue engineering construct. Preliminary work on primary animal neuronal cells in response to chemoattractant and topographic cue within the scaffolds has produced encouraging results.

  18. Biological Atomic Force Microscopy for Imaging Gold-Labeled Liposomes on Human Coronary Artery Endothelial Cells

    PubMed Central

    Zaske, Ana-María; Danila, Delia; Queen, Michael C.; Golunski, Eva; Conyers, Jodie L.

    2013-01-01

    Although atomic force microscopy (AFM) has been used extensively to characterize cell membrane structure and cellular processes such as endocytosis and exocytosis, the corrugated surface of the cell membrane hinders the visualization of extracellular entities, such as liposomes, that may interact with the cell. To overcome this barrier, we used 90 nm nanogold particles to label FITC liposomes and monitor their endocytosis on human coronary artery endothelial cells (HCAECs) in vitro. We were able to study the internalization process of gold-coupled liposomes on endothelial cells, by using AFM. We found that the gold-liposomes attached to the HCAEC cell membrane during the first 15–30 min of incubation, liposome cell internalization occurred from 30 to 60 min, and most of the gold-labeled liposomes had invaginated after 2 hr of incubation. Liposomal uptake took place most commonly at the periphery of the nuclear zone. Dynasore monohydrate, an inhibitor of endocytosis, obstructed the internalization of the gold-liposomes. This study showed the versatility of the AFM technique, combined with fluorescent microscopy, for investigating liposome uptake by endothelial cells. The 90 nm colloidal gold nanoparticles proved to be a noninvasive contrast agent that efficiently improves AFM imaging during the investigation of biological nanoprocesses. PMID:26555999

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

    PubMed Central

    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

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

    NASA Astrophysics Data System (ADS)

    Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei

    2014-09-01

    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.

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

    SciTech Connect

    Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei

    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.

  2. Genetic code expansion enables live-cell and super-resolution imaging of site-specifically labeled cellular proteins.

    PubMed

    Uttamapinant, Chayasith; Howe, Jonathan D; Lang, Kathrin; Beránek, Václav; Davis, Lloyd; Mahesh, Mohan; Barry, Nicholas P; Chin, Jason W

    2015-04-15

    Methods to site-specifically and densely label proteins in cellular ultrastructures with small, bright, and photostable fluorophores would substantially advance super-resolution imaging. Recent advances in genetic code expansion and bioorthogonal chemistry have enabled the site-specific labeling of proteins. However, the efficient incorporation of unnatural amino acids into proteins and the specific, fluorescent labeling of the intracellular ultrastructures they form for subdiffraction imaging has not been accomplished. Two challenges have limited progress in this area: (i) the low efficiency of unnatural amino acid incorporation that limits labeling density and therefore spatial resolution and (ii) the uncharacterized specificity of intracellular labeling that will define signal-to-noise, and ultimately resolution, in imaging. Here we demonstrate the efficient production of cystoskeletal proteins (β-actin and vimentin) containing bicyclo[6.1.0]nonyne-lysine at genetically defined sites. We demonstrate their selective fluorescent labeling with respect to the proteome of living cells using tetrazine-fluorophore conjugates, creating densely labeled cytoskeletal ultrastructures. STORM imaging of these densely labeled ultrastructures reveals subdiffraction features, including nuclear actin filaments. This work enables the site-specific, live-cell, fluorescent labeling of intracellular proteins at high density for super-resolution imaging of ultrastructural features within cells.

  3. Genetic Code Expansion Enables Live-Cell and Super-Resolution Imaging of Site-Specifically Labeled Cellular Proteins

    PubMed Central

    2015-01-01

    Methods to site-specifically and densely label proteins in cellular ultrastructures with small, bright, and photostable fluorophores would substantially advance super-resolution imaging. Recent advances in genetic code expansion and bioorthogonal chemistry have enabled the site-specific labeling of proteins. However, the efficient incorporation of unnatural amino acids into proteins and the specific, fluorescent labeling of the intracellular ultrastructures they form for subdiffraction imaging has not been accomplished. Two challenges have limited progress in this area: (i) the low efficiency of unnatural amino acid incorporation that limits labeling density and therefore spatial resolution and (ii) the uncharacterized specificity of intracellular labeling that will define signal-to-noise, and ultimately resolution, in imaging. Here we demonstrate the efficient production of cystoskeletal proteins (β-actin and vimentin) containing bicyclo[6.1.0]nonyne-lysine at genetically defined sites. We demonstrate their selective fluorescent labeling with respect to the proteome of living cells using tetrazine-fluorophore conjugates, creating densely labeled cytoskeletal ultrastructures. STORM imaging of these densely labeled ultrastructures reveals subdiffraction features, including nuclear actin filaments. This work enables the site-specific, live-cell, fluorescent labeling of intracellular proteins at high density for super-resolution imaging of ultrastructural features within cells. PMID:25831022

  4. Label-free single-cell protein quantification using a drop-based mix-and-read system

    PubMed Central

    Abbaspourrad, Alireza; Zhang, Huidan; Tao, Ye; Cui, Naiwen; Asahara, Haruichi; Zhou, Ying; Yue, Dongxian; Koehler, Stephan A.; Ung, Lloyd W.; Heyman, John; Ren, Yukun; Ziblat, Roy; Chong, Shaorong; Weitz, David A.

    2015-01-01

    Quantitative protein analysis of single cells is rarely achieved due to technical difficulties of detecting minute amounts of proteins present in one cell. We develop a mix-and-read assay for drop-based label-free protein analysis of single cells. This high-throughput method quantifies absolute, rather than relative, amounts of proteins and does not involve antibody labeling or mass spectrometry. PMID:26234416

  5. A new metabolic cell-wall labelling method reveals peptidoglycan in Chlamydia trachomatis.

    PubMed

    Liechti, G W; Kuru, E; Hall, E; Kalinda, A; Brun, Y V; VanNieuwenhze, M; Maurelli, A T

    2014-02-27

    Peptidoglycan (PG), an essential structure in the cell walls of the vast majority of bacteria, is critical for division and maintaining cell shape and hydrostatic pressure. Bacteria comprising the Chlamydiales were thought to be one of the few exceptions. Chlamydia harbour genes for PG biosynthesis and exhibit susceptibility to 'anti-PG' antibiotics, yet attempts to detect PG in any chlamydial species have proven unsuccessful (the 'chlamydial anomaly'). We used a novel approach to metabolically label chlamydial PG using d-amino acid dipeptide probes and click chemistry. Replicating Chlamydia trachomatis were labelled with these probes throughout their biphasic developmental life cycle, and the results of differential probe incorporation experiments conducted in the presence of ampicillin are consistent with the presence of chlamydial PG-modifying enzymes. These findings culminate 50 years of speculation and debate concerning the chlamydial anomaly and are the strongest evidence so far that chlamydial species possess functional PG.

  6. Radionuclide-labeled red blood cell imaging of vascular malformations in children

    SciTech Connect

    Sloan, G.M.; Bolton, L.L.; Miller, J.H.; Reinisch, J.F.; Nichter, L.S.

    1988-09-01

    Vascular malformations, particularly in the absence of cutaneous changes, can be difficult to distinguish from other soft tissue masses in children. We have used technetium-99m-labeled red blood cell scintigraphy to study 47 lesions in 43 children. Thirty-nine lesions showed increased flow and were, therefore, diagnosed as vascular malformations. Subsequent biopsy of 10 of these lesions confirmed that diagnosis. The other 29 lesions with increased flow were followed for 10 months to 5 years and the clinical course was consistent with vascular malformation in every case. Eight lesions showed no increased flow on technetium scan. One of these subsequently proved to be a hemangioma. The others have turned out not to be vascular malformations. Therefore, in our experience, the technetium-99m-labeled red blood cell scan has had 98% sensitivity and 100% specificity in diagnosing vascular malformations in children.

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

    PubMed Central

    Yoon, Jonghee; Kim, Kyoohyun; 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. PMID:26504637

  8. Photoaffinity labeling of regulatory subunits of protein kinase A in cardiac cell fractions of rats

    NASA Technical Reports Server (NTRS)

    Mednieks, M. I.; Popova, I.; Grindeland, R. E.

    1992-01-01

    Photoaffinity labeling in heart tissue of rats flown on Cosmos 2044 was used to measure the regulatory (R) subunits of adenosine monophosphate-dependent protein kinase. A significant decrease of RII subunits in the particulate cell fraction extract (S2; P less than 0.05 in all cases) was observed when extracts of tissue samples from vivarium controls were compared with those from flight animals. Photoaffinity labeling of the soluble fraction (S1) was observed to be unaffected by spaceflight or any of the simulation conditions. Proteins of the S2 fraction constitute a minor (less than 10 percent) component of the total, whereas the S1 fraction contained most of the cell proteins. Changes in a relatively minor aspect of adenosine monophosphate-mediated reactions are considered to be representative of a metabolic effect.

  9. Manganese-impregnated mesoporous silica nanoparticles for signal enhancement in MRI cell labelling studies

    NASA Astrophysics Data System (ADS)

    Guillet-Nicolas, Rémy; Laprise-Pelletier, Myriam; Nair, Mahesh M.; Chevallier, Pascale; Lagueux, Jean; Gossuin, Yves; Laurent, Sophie; Kleitz, Freddy; Fortin, Marc-André

    2013-11-01

    Mesoporous silica nanoparticles (MSNs) are used in drug delivery and cell tracking applications. As Mn2+ is already implemented as a ``positive'' cell contrast agent in preclinical imaging procedures (in the form of MnCl2 for neurological studies), the introduction of Mn in the porous network of MSNs would allow labelling cells and tracking them using MRI. These particles are in general internalized in endosomes, an acidic environment with high saline concentration. In addition, the available MSN porosity could also serve as a carrier to deliver medical/therapeutic substances through the labelled cells. In the present study, manganese oxide was introduced in the porous network of MCM-48 silica nanoparticles (Mn-M48SNs). The particles exhibit a narrow size distribution (~140 nm diam.) and high porosity (~60% vol.), which was validated after insertion of Mn. The resulting Mn-M48SNs were characterized by TEM, N2 physisorption, and XRD. Evidence was found with H2-TPR, and XPS characterization, that Mn(ii) is the main oxidation state of the paramagnetic species after suspension in water, most probably in the form of Mn-OOH. The colloidal stability as a function of time was confirmed by DLS in water, acetate buffer and cell culture medium. In NMR data, no significant evidence of Mn2+ leaching was found in Mn-M48SNs in acidic water (pH 6), up to 96 hours after suspension. High longitudinal relaxivity values of r1 = 8.4 mM-1 s-1 were measured at 60 MHz and 37 °C, with the lowest relaxometric ratios (r2/r1 = 2) reported to date for a Mn-MSN system. Leukaemia cells (P388) were labelled with Mn-M48SNs and nanoparticle cell internalization was confirmed by TEM. Finally, MRI contrast enhancement provided by cell labelling with escalated incubation concentrations of Mn-M48SNs was quantified at 1 T. This study confirmed the possibility of efficiently confining Mn into M48SNs using incipient wetness, while maintaining an open porosity and relatively high pore volume. Because

  10. Label-free magnetic resonance imaging to locate live cells in three-dimensional porous scaffolds

    PubMed Central

    Abarrategi, A.; Fernandez-Valle, M. E.; Desmet, T.; Castejón, D.; Civantos, A.; Moreno-Vicente, C.; Ramos, V.; Sanz-Casado, J. V.; Martínez-Vázquez, F. J.; Dubruel, P.; Miranda, P.; López-Lacomba, J. L.

    2012-01-01

    Porous scaffolds are widely tested materials used for various purposes in tissue engineering. A critical feature of a porous scaffold is its ability to allow cell migration and growth on its inner surface. Up to now, there has not been a method to locate live cells deep inside a material, or in an entire structure, using real-time imaging and a non-destructive technique. Herein, we seek to demonstrate the feasibility of the magnetic resonance imaging (MRI) technique as a method to detect and locate in vitro non-labelled live cells in an entire porous material. Our results show that the use of optimized MRI parameters (4.7 T; repetition time = 3000 ms; echo time = 20 ms; resolution 39 × 39 µm) makes it possible to obtain images of the scaffold structure and to locate live non-labelled cells in the entire material, with a signal intensity higher than that obtained in the culture medium. In the current study, cells are visualized and located in different kinds of porous scaffolds. Moreover, further development of this MRI method might be useful in several three-dimensional biomaterial tests such as cell distribution studies, routine qualitative testing methods and in situ monitoring of cells inside scaffolds. PMID:22442095

  11. Linear patterning of magnetically labeled Dictyostelium cells to display confined development

    NASA Astrophysics Data System (ADS)

    Frasca, Guillaume; Raynaud, Franck; Bacri, Jean-Claude; Gazeau, Florence; Wilhelm, Claire

    2008-05-01

    In severe nutriment conditions, the social amoeba Dictyostelium discoideum enters a particular life cycle where it forms multicellular patterns to achieve aggregation. Extensively observed from an initial dispersed state, its developmental program can usefully be studied from a confined population to implement theoretical developments regarding biological self-organization. The challenge is then to form a cell assembly of well-defined geometrical dimensions without hindering cell behavior. To achieve this goal, we imposed transient constraints by applying temporary external magnetic gradients to trap magnetically labeled cells. Deposits of various numbers of cells were geometrically characterized for different magnetic exposure conditions. We demonstrated that the cell deposit was organized as a three-dimensional (3D) structure by both stacking layers of cells and extending these layers in the substrate plane. This structure evolves during the aggregation phase, forming periodic aggregative centers along the linear initial pattern.

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

    PubMed

    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.

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

    PubMed

    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

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

    PubMed Central

    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

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

    SciTech Connect

    Sanders, C.L.; Lauhala, K.E.; McDonald, K.E. )

    1989-09-01

    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.

  16. Human aortic endothelial cell labeling with positive contrast gadolinium oxide nanoparticles for cellular magnetic resonance imaging at 7 Tesla.

    PubMed

    Loai, Yasir; Sakib, Nurus; Janik, Rafal; Foltz, Warren D; Cheng, Hai-Ling Margaret

    2012-04-01

    Positive T₁ contrast using gadolinium (Gd) contrast agents can potentially improve detection of labeled cells on magnetic resonance imaging (MRI). Recently, gadolinium oxide (Gd₂O₃) nanoparticles have shown promise as a sensitive T₁ agent for cell labeling at clinical field strengths compared to conventional Gd chelates. The objective of this study was to investigate Gado CELLTrack, a commercially available Gd₂O₃ nanoparticle, for cell labeling and MRI at 7 T. Relaxivity measurements yielded r1  =  4.7 s⁻¹ mM⁻¹ and r₂/r₁  =  6.2. Human aortic endothelial cells were labeled with Gd₂O₃ at various concentrations and underwent MRI from 1 to 7 days postlabeling. The magnetic resonance relaxation times T₁ and T₂ of labeled cell pellets were measured. Cellular contrast agent uptake was quantified by inductively coupled plasma-atomic emission spectroscopy, which showed very high uptake compared to conventional Gd compounds. MRI demonstrated significant positive T₁ contrast and stable labeling on cells. Enhancement was optimal at low Gd concentrations, attained in the 0.02 to 0.1 mM incubation concentration range (corresponding cell uptake was 7.26 to 34.1 pg Gd/cell). Cell viability and proliferation were unaffected at the concentrations tested and up to at least 3 days postlabeling. Gd₂O₃ is a promising sensitive and stable positive contrast agent for cellular MRI at 7 T.

  17. Poly(L-lysine)-modified iron oxide nanoparticles for stem cell labeling.

    PubMed

    Babic, Michal; Horák, Daniel; Trchová, Miroslava; Jendelová, Pavla; Glogarová, Katerina; Lesný, Petr; Herynek, Vít; Hájek, Milan; Syková, Eva

    2008-03-01

    New surface-modified iron oxide nanoparticles were developed by precipitation of Fe(II) and Fe(III) salts with ammonium hydroxide and oxidation of the resulting magnetite with sodium hypochlorite, followed by the addition of poly( L-lysine) (PLL) solution. PLL of several molecular weights ranging from 146 ( L-lysine) to 579 000 was tested as a coating to boost the intracellular uptake of the nanoparticles. The nanoparticles were characterized by TEM, dynamic light scattering, FTIR, and ultrasonic spectrometry. TEM revealed that the particles were ca. 6 nm in diameter, while FTIR showed that their surfaces were well-coated with PLL. The interaction of PLL-modified iron oxide nanoparticles with DMEM culture medium was verified by UV-vis spectroscopy. Rat bone marrow stromal cells (rMSCs) and human mesenchymal stem cells (hMSC) were labeled with PLL-modified iron oxide nanoparticles or with Endorem (control). Optical microscopy and TEM confirmed the presence of PLL-modified iron oxide nanoparticles inside the cells. Cellular uptake was very high (more than 92%) for PLL-modified nanoparticles that were coated with PLL (molecular weight 388 00) at a concentration of 0.02 mg PLL per milliliter of colloid. The cellular uptake of PLL-modified iron oxide was facilitated by its interaction with the negatively charged cell surface and subsequent endosomolytic uptake. The relaxivity of rMSCs labeled with PLL-modified iron oxide and the amount of iron in the cells were determined. PLL-modified iron oxide-labeled rMSCs were imaged in vitro and in vivo after intracerebral grafting into the contralateral hemisphere of the adult rat brain. The implanted cells were visible on magnetic resonance (MR) images as a hypointense area at the injection site and in the lesion. In comparison with Endorem, nanoparticles modified with PLL of an optimum molecular weight demonstrated a higher efficiency of intracellular uptake by MSC cells.

  18. Label-Retaining, Quiescent Globose Basal Cells Are Found in the Olfactory Epithelium

    PubMed Central

    Jang, Woochan; Chen, Xueyan; Flis, Daniel; Harris, Margaret; Schwob, James E.

    2014-01-01

    The vertebrate olfactory epithelium (OE) is known for its ability to renew itself throughout life as well as to reconstitute after injury. Although this remarkable capacity demonstrates the persistence of stem cells and multipotent progenitor cells, their nature in the OE remains undefined and controversial, as both horizontal basal cells (HBCs) and globose basal cells (GBCs) have features in common with each other and with stem cells in other tissues. Here, we investigate whether some among the population of GBCs satisfy a key feature of stem cells, i.e., mitotic quiescence with retention of thymidine analogue label and activation by injury. Accordingly, we demonstrate that some GBCs express p27Kip1, a member of the Kip/Cip family of cyclin-dependent kinase inhibitors. In addition, some GBCs retain bromodeoxyuridine or ethynyldeoxyuridine for an extended period when the pulse is administered in neonates followed by a 1-month chase. Their identity as GBCs was confirmed by electron microscopy. All spared GBCs express Ki-67 in the methyl bromide (MeBr)-lesioned OE initially after lesion, indicating that the label-retaining (LR) GBCs are activated in response to injury. LR-GBCs reappear during the acute recovery period following MeBr exposure, as demonstrated with 2- or 4-week chase periods after labeling. Taken together, our data demonstrate the existence of LR-GBCs that are seemingly activated in response to epithelial injury and then re-established after the initial phase of recovery is completed. In this regard, some among the GBCs satisfy a common criterion for functioning like stem cells. PMID:24122672

  19. High-Throughput, Single-Cell Analysis of Macrophage Interactions with Fluorescently Labeled Bacillus anthracis Spores▿

    PubMed Central

    Stojkovic, Bojana; Torres, Eric M.; Prouty, Angela M.; Patel, Hetal K.; Zhuang, Lefan; Koehler, Theresa M.; Ballard, Jimmy D.; Blanke, Steven R.

    2008-01-01

    The engulfment of Bacillus anthracis spores by macrophages is an important step in the pathogenesis of inhalational anthrax. However, from a quantitative standpoint, the magnitude to which macrophages interact with and engulf spores remains poorly understood, in part due to inherent limitations associated with commonly used assays. To analyze phagocytosis of spores by RAW264.7 macrophage-like cells in a high-throughput, nonsubjective manner, we labeled B. anthracis Sterne 7702 spores prior to infection with an Alexa Fluor 488 amine-reactive dye in a manner that did not alter their germination, growth kinetics, and heat resistance. Using flow cytometry, large numbers of cells exposed to labeled spores were screened to concurrently discriminate infected from uninfected cells and surface-associated from internalized spores. These experiments revealed that spore uptake was not uniform, but instead, highly heterogeneous and characterized by subpopulations of infected and uninfected cells, as well as considerable variation in the number of spores associated with individual cells. Flow cytometry analysis of infections demonstrated that spore uptake was independent of the presence or absence of fetal bovine serum, a germinant that, while routinely used in vitro, complicates the interpretation of the outcome of infections. Two commonly used macrophage cell lines, RAW264.7 and J774A.1 cells, were compared, revealing significant disparity between these two models in the rates of phagocytosis of labeled spores. These studies provide the experimental framework for investigating mechanisms of spore phagocytosis, as well as quantitatively evaluating strategies for interfering with macrophage binding and uptake of spores. PMID:18552183

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  2. Imaging viral RNA using multiply labeled tetravalent RNA imaging probes in live cells.

    PubMed

    Alonas, Eric; Vanover, Daryll; Blanchard, Emmeline; Zurla, Chiara; Santangelo, Philip J

    2016-04-01

    Viruses represent an important class of pathogens that have had an enormous impact on the health of the human race. They are extraordinarily diverse; viral particles can range in size from ∼80nm to ∼10μm in length, and contain genomes with RNA or DNA strands. Regardless of their genome type, RNA species are frequently generated as a part of their replication process, and for viruses with RNA genomes, their loading into the virion represents a critical step in the creation of infectious particles. RNA imaging tools represent a powerful approach to gain insight into fundamental viral processes, including virus entry, replication, and virion assembly. Imaging viral processes in live cells is critical due to both the heterogeneity of these processes on a per cell basis, and the inherent dynamics of these processes. There are a number of methods for labeling RNA in live cells; we'll introduce the myriad of methods and then focus on one approach for labeling viral RNA, using multiply-labeled tetravalent RNA imaging probes (MTRIPs), which do not require engineering of the target RNAs. We feel this approach is advantageous given many viral genomes may not tolerate large nucleotide insertions into their sequences. PMID:26875782

  3. Indium 111-labeled white blood cell scans after vascular prosthetic reconstruction

    SciTech Connect

    Sedwitz, M.M.; Davies, R.J.; Pretorius, H.T.; Vasquez, T.E.

    1987-11-01

    The clinical value of indium 111-labeled white blood cell (WBC) scanning done after vascular graft procedures was investigated to differentiate noninfectious postoperative inflammation associated with graft incorporation from early prosthetic graft infection. Indium 111-labeled WBC scans were initially obtained in 30 patients before discharge from the hospital and during the subsequent follow-up period (334 days). Fourteen of 30 patients (47%) had normal predischarge scans that included all 10 patients who had grafts confined to the abdomen and 4 of 20 patients (20%) who had grafts arising or terminating at the femoral arteries (p less than 0.05). Sixteen of 30 patients (53%) discharged with abnormal initial indium 111 WBC scans underwent serial scanning until the scan normalized or a graft complication developed. All of the 16 patients had grafts involving the groin region. Abnormal indium 111 uptake in the femoral region continued for a mean 114 days without the development of prosthetic graft infections. The sensitivity of indium 111-labeled WBC scans for detecting wound complications was 100%, whereas the specificity was 50%. Thus, the accuracy of the test was only 53%. We conclude that (1) abnormal indium 111 WBC scans are common after graft operations involving the groin region but are unusual after vascular procedures confined to the abdomen, and (2) in the absence of clinical suspicion, the indium 111-labeled WBC scan does not reliably predict prosthetic graft infection because of the low specificity of the test in the early postoperative period.

  4. In-vitro Optimization of Nanoparticle-Cell Labeling Protocols for In-vivo Cell Tracking Applications

    PubMed Central

    Betzer, Oshra; Meir, Rinat; Dreifuss, Tamar; Shamalov, Katerina; Motiei, Menachem; Shwartz, Amit; Baranes, Koby; Cohen, Cyrille J.; Shraga-Heled, Niva; Ofir, Racheli; Yadid, Gal; Popovtzer, Rachela

    2015-01-01

    Recent advances in theranostic nanomedicine can promote stem cell and immune cell-based therapy. Gold nanoparticles (GNPs) have been shown to be promising agents for in-vivo cell-tracking in cell-based therapy applications. Yet a crucial challenge is to develop a reliable protocol for cell upload with, on the one hand, sufficient nanoparticles to achieve maximum visibility of cells, while on the other hand, assuring minimal effect of particles on cell function and viability. Previous studies have demonstrated that the physicochemical parameters of GNPs have a critical impact on their efficient uptake by cells. In the current study we have examined possible variations in GNP uptake, resulting from different incubation period and concentrations in different cell-lines. We have found that GNPs effectively labeled three different cell-lines - stem, immune and cancer cells, with minimal impairment to cell viability and functionality. We further found that uptake efficiency of GNPs into cells stabilized after a short period of time, while GNP concentration had a significant impact on cellular uptake, revealing cell-dependent differences. Our results suggest that while heeding the slight variations within cell lines, modifying the loading time and concentration of GNPs, can promote cell visibility in various nanoparticle-dependent in-vivo cell tracking and imaging applications. PMID:26507853

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

    PubMed

    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. PMID:27305814

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

    PubMed

    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.

  7. On-chip integrated labelling, transport and detection of tumour cells.

    PubMed

    Woods, Jane; Docker, Peter T; Dyer, Charlotte E; Haswell, Stephen J; Greenman, John

    2011-11-01

    Microflow cytometry represents a promising tool for the investigation of diagnostic and prognostic cellular cancer markers, particularly if integrated within a device that allows primary cells to be freshly isolated from the solid tumour biopsies that more accurately reflect patient-specific in vivo tissue microenvironments at the time of staining. However, current tissue processing techniques involve several sequential stages with concomitant cell losses, and as such are inappropriate for use with small biopsies. Accordingly, we present a simple method for combined antibody-labelling and dissociation of heterogeneous cells from a tumour mass, which reduces the number of processing steps. Perfusion of ex vivo tissue at 4°C with antibodies and enzymes slows cellular activity while allowing sufficient time for the diffusion of minimally active enzymes. In situ antibody-labelled cells are then dissociated at 37°C from the tumour mass, whereupon hydrogel-filled channels allow the release of relatively low cell numbers (<1000) into a biomimetic microenvironment. This novel approach to sample processing is then further integrated with hydrogel-based electrokinetic transport of the freshly liberated fluorescent cells for downstream detection. It is anticipated that this integrated microfluidic methodology will have wide-ranging biomedical and clinical applications.

  8. 2D light scattering static cytometry for label-free single cell analysis with submicron resolution.

    PubMed

    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.

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

    PubMed Central

    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

  10. Optimized tools for multicolor stochastic labeling reveal diverse stereotyped cell arrangements in the fly visual system

    PubMed Central

    Nern, Aljoscha; Pfeiffer, Barret D.; Rubin, Gerald M.

    2015-01-01

    We describe the development and application of methods for high-throughput neuroanatomy in Drosophila using light microscopy. These tools enable efficient multicolor stochastic labeling of neurons at both low and high densities. Expression of multiple membrane-targeted and distinct epitope-tagged proteins is controlled both by a transcriptional driver and by stochastic, recombinase-mediated excision of transcription-terminating cassettes. This MultiColor FlpOut (MCFO) approach can be used to reveal cell shapes and relative cell positions and to track the progeny of precursor cells through development. Using two different recombinases, the number of cells labeled and the number of color combinations observed in those cells can be controlled separately. We demonstrate the utility of MCFO in a detailed study of diversity and variability of Distal medulla (Dm) neurons, multicolumnar local interneurons in the adult visual system. Similar to many brain regions, the medulla has a repetitive columnar structure that supports parallel information processing together with orthogonal layers of cell processes that enable communication between columns. We find that, within a medulla layer, processes of the cells of a given Dm neuron type form distinct patterns that reflect both the morphology of individual cells and the relative positions of their arbors. These stereotyped cell arrangements differ between cell types and can even differ for the processes of the same cell type in different medulla layers. This unexpected diversity of coverage patterns provides multiple independent ways of integrating visual information across the retinotopic columns and implies the existence of multiple developmental mechanisms that generate these distinct patterns. PMID:25964354

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

    NASA Astrophysics Data System (ADS)

    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.

  12. Macrophage recognition of immune complexes: development and application of novel cell surface labeling procedures.

    PubMed

    Petty, H R; Dereski, W

    1985-07-16

    A fluorescein- and lactoperoxidase-conjugated ferritin-anti-ferritin immune complex has been prepared for cell surface labeling experiments on immune recognition and effector function. Lactoperoxidase (LPO) has been covalently coupled to affinity-purified anti-ferritin antibodies with p-benzoquinone by a modified version of the method of Ternynck and Avrameas [Ternynck, T., & Avrameas, S. (1976) Ann. Immunol. (Paris) 127C, 197]. The conjugate is a heterodimer of Mr230 000 with linkages to either or both of the heavy and light chains of the antibody, as judged by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence and presence of 2-mercaptoethanol. The conjugate retains antibody-binding activity as measured by a quantitative precipitin assay. When incorporated into immune complexes, the modified antibody also retains Fc receptor recognition ability as determined by erythrocyte-antibody rosette inhibition assays. Electron microscopy demonstrated that the antigen, ferritin, was monodisperse with complete apoprotein sheaths surrounding the core. Ferritin-anti-ferritin-LPO complexes were formed in 4-fold antigen excess. Complexes were verified by fluorescence and electron microscopy. Immune complexes were masked with "cold" iodine by use of the endogenous LPO activity. The complexes bound to cells at 4 degrees C as shown by electron microscopy and fluorescence video/intensification microscopy. The LPO delivered to the cell surface in this fashion can be utilized to iodinate the surface with 125I. Under saturation conditions, the labeling with local LPO delivery followed by SDS-PAGE and autoradiography is identical with labeling with free LPO. Labeling has also been conducted under conditions of substrate deficit.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:4052386

  13. Ptychography – a label free, high-contrast imaging technique for live cells using quantitative phase information

    PubMed Central

    Marrison, Joanne; Räty, Lotta; Marriott, Poppy; O'Toole, Peter

    2013-01-01

    Cell imaging often relies on synthetic or genetic fluorescent labels, to provide contrast which can be far from ideal for imaging cells in their in vivo state. We report on the biological application of a, label-free, high contrast microscopy technique known as ptychography, in which the image producing step is transferred from the microscope lens to a high-speed phase retrieval algorithm. We demonstrate that this technology is appropriate for label-free imaging of adherent cells and is particularly suitable for reporting cellular changes such as mitosis, apoptosis and cell differentiation. The high contrast, artefact-free, focus-free information rich images allow dividing cells to be distinguished from non-dividing cells by a greater than two-fold increase in cell contrast, and we demonstrate this technique is suitable for downstream automated cell segmentation and analysis. PMID:23917865

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

    PubMed

    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.

  15. Degradation of 14C-labeled streptococcal cell walls by egg white lysozyme and lysosomal enzymes.

    PubMed Central

    Gallis, H A; Miller, S E; Wheat, R W

    1976-01-01

    The resistance of native and trypsin-treated [14C] glucose-labeled cell walls to degradation by lysozyme and human lysosomal enzymes was confirmed. In contrast, chemically N-acetylated cell walls undergo significant degradation by these enzymes in the pH range of 4.5 to 5.5 without prior removal of the group-specific carbohydrate. N-acetylation after removal of the group A carbohydrate by formamide extraction renders the cell walls considerably more susceptible to these enzymes than by formamaide extraction alone. It appears, therefore, that unless N-acetylation can occur in vivo, streptococcal cell walls are minimally degraded, if at all, by human peripheral blood leukocytes or lysozyme. Examination of leukocyte extracts from normal subjects and patients with post-streptococcal syndromes revealed no qualitative differences in ability to dissolve streptococcal cell walls. Images PMID:773836

  16. Efficient (18)F-Labeling of Synthetic Exendin-4 Analogues for Imaging Beta Cells.

    PubMed

    Keliher, Edmund J; Reiner, Thomas; Thurber, Greg M; Upadhyay, Rabi; Weissleder, Ralph

    2012-08-01

    A number of exendin derivatives have been developed to target glucagon-like peptide 1 (GLP-1) receptors on beta cells in vivo. Modifications of exendin analogues have been shown to have significant effects on pharmacokinetics and, as such, have been used to develop a variety of therapeutic compounds. Here, we show that an exendin-4, modified at position 12 with a cysteine conjugated to a tetrazine, can be labeled with (18)F-trans-cyclooctene and converted into a PET imaging agent at high yields and with good selectivity. The agent accumulates in beta cells in vivo and has sufficiently high accumulation in mouse models of insulinomas to enable in vivo imaging.

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

    SciTech Connect

    Neft, R.E.; Tierney, L.A.; Belinsky, S.A.

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  19. Label-free imaging of goblet cells as a marker for differentiating colonic polyps by multiphoton microscopy Label-free imaging of goblet cells

    NASA Astrophysics Data System (ADS)

    Zhuo, S. M.; Wu, G. Z.; Chen, J. X.; Zhu, X. Q.; Xie, S. S.

    2012-06-01

    Discrimination of adenomas from hyperplastic polyps can reduce the risk of unnecessary complications and healthcare cost. However, it is challenging during colonoscopy screening, and histological analysis remains the ``gold standard'' for the final diagnosis. Here, we describe a label-free imaging method, multiphoton microscopy (MPM), to the discrimination between adenomas and hyperplastic polyps. We find that multiphoton imaging provides cellular and subcellular details to the identification of adenomas from hyperplastic polyps. In particular, there is significant difference in the population density of goblet cells among normal colon, hyperplastic polyp, and adenoma, providing substantial potential to become a quantitative intrinsic marker for in vivo clinical diagnosis of early colonic lesions. To our knowledge, this is the first demonstration of the potential of MPM for differentiation of colonic polyps.

  20. Temperature-induced labelling of Fluo-3 AM selectively yields brighter nucleus in adherent cells

    SciTech Connect

    Meng, Guixian; Pan, Leiting; Li, Cunbo; Hu, Fen; Shi, Xuechen; Lee, Imshik; Drevenšek-Olenik, Irena; Zhang, Xinzheng; Xu, Jingjun

    2014-01-17

    Highlights: •We detailedly examine temperature effects of Fluo-3 AM labelling in adherent cells. •4 °C Loading and 20 °C de-esterification of Fluo-3 AM yields brighter nuclei. •Brighter nuclei labelling by Fluo-3 AM also depends on cell adhesion quality. •A qualitative model of the brighter nucleus is proposed. -- Abstract: Fluo-3 is widely used to study cell calcium. Two traditional approaches: (1) direct injection and (2) Fluo-3 acetoxymethyl ester (AM) loading, often bring conflicting results in cytoplasmic calcium ([Ca{sup 2+}]{sub c}) and nuclear calcium ([Ca{sup 2+}]{sub n}) imaging. AM loading usually yields a darker nucleus than in cytoplasm, while direct injection always induces a brighter nucleus which is more responsive to [Ca{sup 2+}]{sub n} detection. In this work, we detailedly investigated the effects of loading and de-esterification temperatures on the fluorescence intensity of Fluo-3 in response to [Ca{sup 2+}]{sub n} and [Ca{sup 2+}]{sub c} in adherent cells, including osteoblast, HeLa and BV2 cells. Interestingly, it showed that fluorescence intensity of nucleus in osteoblast cells was about two times larger than that of cytoplasm when cells were loaded with Fluo-3 AM at 4 °C and allowed a subsequent step for de-esterification at 20 °C. Brighter nuclei were also acquired in HeLa and BV2 cells using the same experimental condition. Furthermore, loading time and adhesion quality of cells had effect on fluorescence intensity. Taken together, cold loading and room temperature de-esterification treatment of Fluo-3 AM selectively yielded brighter nucleus in adherent cells.

  1. Optofluidic device for label-free cell classification from whole blood.

    PubMed

    Wu, Tsung-Feng; Lo, Yu-Hwa

    2015-01-01

    A unique optofluidic lab-on-a-chip device that can detect optically encoded forward scattering signals is demonstrated. With a unique design of a spatial mask that patterns the intensity distribution of the illuminating light, the position and velocity of each travelling cell in the flow can be measured with submicrometer resolution, which enables the generation of a cell distribution plot over the cross section of the channel. The distribution of cells is highly sensitive to its size and stiffness, both being important biomarkers for cell classification without cell labelling. The optical-coding technique offers an easy route to classify cells based on their size and stiffness. Because the stiffness and size of neutrophils are distinct from other types of white blood cells, the number of neutrophils can be detected from other white blood cells and red blood cells. Above all, the enumeration of neutrophil concentration can be obtained from only 5 μL of human blood with a simple blood preparation process saving the usual steps of anticoagulation, centrifugation, antibody labelling, or filtering. The optofluidic system is compact, inexpensive, and simple to fabricate and operate. The system uses a commodity laser diode and a Si PIN photoreceiver and digital signal processing to extract vital information about cells and suppress the noise from the encoded optical scattering signals. The optofluidic device holds promise to be a point-of-care and home care device to measure neutrophil concentration, which is the key indicator of the immune functions for cancer patients undergoing chemotherapy. PMID:25626540

  2. A New Method for Preparing Mesenchymal Stem Cells and Labeling with Ferumoxytol for Cell Tracking by MRI

    PubMed Central

    Liu, Li; Tseng, Lanya; Ye, Qing; Wu, Yijen L.; Bain, Daniel J.; Ho, Chien

    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 MSCs to prepare native MSCs, so that (i) the phagocytic activity of cultured MSCs can be recovered and expanded MSCs can be ex-vivo labeled with Ferumoxytol, which is currently the only FDA approved SPIO nanoparticles for human use. Using our new method, 7-day cultured MSCs regain the capability to take up Ferumoxytol and exhibit an intracellular iron concentration of 2.50 ± 0.50 pg/MSC, comparable to that obtained by using Ferumoxytol-heparin-protamine nanocomplex; and (ii) cells can be re-sized to more native size, reducing from 32.0 ± 7.2 μm to 19.5 ± 5.2 μm. Our method can be very useful for expanding MSCs and labeling with Ferumoxytol, without the need for transfection agents and/or electroporation, allowing cell-tracking by MRI in both pre-clinical and clinical studies. PMID:27188664

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

    SciTech Connect

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  5. Single molecule tracking of quantum dot-labeled mRNAs in a cell nucleus

    SciTech Connect

    Ishihama, Yo; Funatsu, Takashi

    2009-03-27

    Single particle tracking (SPT) is a powerful technique for studying mRNA dynamics in cells. Although SPT of mRNA has been performed by labeling mRNA with fluorescent dyes or proteins, observation of mRNA for long durations with high temporal resolution has been difficult due to weak fluorescence and rapid photobleaching. Using quantum dots (QDs), we succeeded in observing the movement of individual mRNAs for more than 60 s, with a temporal resolution of 30 ms. Intronless and truncated ftz mRNA, synthesized in vitro and labeled with QDs, was microinjected into the nuclei of Cos7 cells. Almost all mRNAs were in motion, and statistical analyses revealed anomalous diffusion between barriers, with a microscopic diffusion coefficient of 0.12 {mu}m{sup 2}/s and a macroscopic diffusion coefficient of 0.025 {mu}m{sup 2}/s. Diffusion of mRNA was observed in interchromatin regions but not in histone2B-GFP-labeled chromatin regions. These results provide direct evidence of channeled mRNA diffusion in interchromatin regions.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

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

    2013-11-29

    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.

  8. Tomographic sensing and localization of fluorescently labeled circulating cells in mice in vivo

    PubMed Central

    Zettergren, Eric; Swamy, Tushar; Runnels, Judith; Lin, Charles P; Niedre, Mark

    2013-01-01

    Sensing and enumeration of specific types of circulating cells in small animals is an important problem in many areas of biomedical research. Microscopy-based fluorescence in vivo flow cytometry methods have been developed previously, but these are typically limited to sampling of very small blood volumes, so that very rare circulating cells may escape detection. Recently, we described the development of a ‘diffuse fluorescence flow cytometer’ (DFFC) that allows sampling of much larger blood vessels and therefore circulating blood volumes in the hindlimb, forelimb or tail of a mouse. In this work, we extend this concept by developing and validating a method to tomographically localize circulating fluorescently labeled cells in the cross section of a tissue simulating optical flow phantom and mouse limb. This was achieved using two modulated light sources and an array of six fiber-coupled detectors that allowed rapid, high-sensitivity acquisition of full tomographic data sets at 10 Hz. These were reconstructed into two-dimensional cross-sectional images using Monte Carlo models of light propagation and the randomized algebraic reconstruction technique. We were able to obtain continuous images of moving cells in the sample cross section with 0.5 mm accuracy or better. We first demonstrated this concept in limb-mimicking optical flow photons with up to four flow channels, and then in the tails of mice with fluorescently labeled multiple myeloma cells. This approach increases the overall diagnostic utility of our DFFC instrument. PMID:22750660

  9. Planar Photonic Crystal Biosensor for Quantitative Label-Free Cell Attachment Microscopy

    PubMed Central

    Chen, Weili; Long, Kenneth D.; Kurniawan, Jonas; Hung, Margaret; Yu, Hojeong; Harley, Brendan A.

    2016-01-01

    In this study, a planar-surface photonic crystal (PC) biosensor for quantitative, kinetic, label-free imaging of cell–surface interactions is demonstrated. The planar biosensor surface eliminates external stimuli to the cells caused by substrate topography to more accurately reflect smooth surface environment encountered by many cell types in vitro. Here, a fabrication approach that combines nanoreplica molding and a horizontal dipping process is used to planarize the surface of the PC biosensor. The planar PC biosensor maintains a high detection sensitivity that enables the monitoring of live cell–substrate interactions with spatial resolution sufficient for observing intracellular attachment strength gradients and the extensions of filopodia from the cell body. The evolution of cell morphology during the attachment and spreading process of 3T3 fibroblast cells is compared between planar and grating-structured PC biosensors. The planar surface effectively eliminates the directionally biased cellular attachment behaviors that are observed on the grating-structured surface. This work represents an important step forward in the development of label-free techniques for observing cellular processes without unintended external environmental modulation. PMID:26877910

  10. Tracking epigenetic histone modifications in single cells using Fab-based live endogenous modification labeling

    PubMed Central

    Hayashi-Takanaka, Yoko; Yamagata, Kazuo; Wakayama, Teruhiko; Stasevich, Timothy J.; Kainuma, Takashi; Tsurimoto, Toshiki; Tachibana, Makoto; Shinkai, Yoichi; Kurumizaka, Hitoshi; Nozaki, Naohito; Kimura, Hiroshi

    2011-01-01

    Histone modifications play an important role in epigenetic gene regulation and genome integrity. It remains largely unknown, however, how these modifications dynamically change in individual cells. By using fluorescently labeled specific antigen binding fragments (Fabs), we have developed a general method to monitor the distribution and global level of endogenous histone H3 lysine modifications in living cells without disturbing cell growth and embryo development. Fabs produce distinct nuclear patterns that are characteristic of their target modifications. H3K27 trimethylation-specific Fabs, for example, are concentrated on inactive X chromosomes. As Fabs bind their targets transiently, the ratio of bound and free molecules depends on the target concentration, allowing us to measure changes in global modification levels. High-affinity Fabs are suitable for mouse embryo imaging, so we have used them to monitor H3K9 and H3K27 acetylation levels in mouse preimplantation embryos produced by in vitro fertilization and somatic cell nuclear transfer. The data suggest that a high level of H3K27 acetylation is important for normal embryo development. As Fab-based live endogenous modification labeling (FabLEM) is broadly useful for visualizing any modification, it should be a powerful tool for studying cell signaling and diagnosis in the future. PMID:21576221

  11. Label-free optical detection of cells grown in 3D silicon microstructures.

    PubMed

    Merlo, Sabina; Carpignano, Francesca; Silva, Gloria; Aredia, Francesca; Scovassi, A Ivana; Mazzini, Giuliano; Surdo, Salvatore; Barillaro, Giuseppe

    2013-08-21

    We demonstrate high aspect-ratio photonic crystals that could serve as three-dimensional (3D) microincubators for cell culture and also provide label-free optical detection of the cells. The investigated microstructures, fabricated by electrochemical micromachining of standard silicon wafers, consist of periodic arrays of silicon walls separated by narrow deeply etched air-gaps (50 μm high and 5 μm wide) and feature the typical spectral properties of photonic crystals in the wavelength range 1.0-1.7 μm: their spectral reflectivity is characterized by wavelength regions where reflectivity is high (photonic bandgaps), separated by narrow wavelength regions where reflectivity is very low. In this work, we show that the presence of cells, grown inside the gaps, strongly affects light propagation across the photonic crystal and, therefore, its spectral reflectivity. Exploiting a label-free optical detection method, based on a fiberoptic setup, we are able to probe the extension of cells adherent to the vertical silicon walls with a non-invasive direct testing. In particular, the intensity ratio at two wavelengths is the experimental parameter that can be well correlated to the cell spreading on the silicon wall inside the gaps.

  12. Tomographic sensing and localization of fluorescently labeled circulating cells in mice in vivo

    NASA Astrophysics Data System (ADS)

    Zettergren, Eric; Swamy, Tushar; Runnels, Judith; Lin, Charles P.; Niedre, Mark

    2012-07-01

    Sensing and enumeration of specific types of circulating cells in small animals is an important problem in many areas of biomedical research. Microscopy-based fluorescence in vivo flow cytometry methods have been developed previously, but these are typically limited to sampling of very small blood volumes, so that very rare circulating cells may escape detection. Recently, we described the development of a ‘diffuse fluorescence flow cytometer’ (DFFC) that allows sampling of much larger blood vessels and therefore circulating blood volumes in the hindlimb, forelimb or tail of a mouse. In this work, we extend this concept by developing and validating a method to tomographically localize circulating fluorescently labeled cells in the cross section of a tissue simulating optical flow phantom and mouse limb. This was achieved using two modulated light sources and an array of six fiber-coupled detectors that allowed rapid, high-sensitivity acquisition of full tomographic data sets at 10 Hz. These were reconstructed into two-dimensional cross-sectional images using Monte Carlo models of light propagation and the randomized algebraic reconstruction technique. We were able to obtain continuous images of moving cells in the sample cross section with 0.5 mm accuracy or better. We first demonstrated this concept in limb-mimicking optical flow photons with up to four flow channels, and then in the tails of mice with fluorescently labeled multiple myeloma cells. This approach increases the overall diagnostic utility of our DFFC instrument.

  13. Using Genetically Encodable Self-Assembling Gd(III) Spin Labels To Make In-Cell Nanometric Distance Measurements.

    PubMed

    Mascali, Florencia C; Ching, H Y Vincent; Rasia, Rodolfo M; Un, Sun; Tabares, Leandro C

    2016-09-01

    Double electron-electron resonance (DEER) can be used to study the structure of a protein in its native cellular environment. Until now, this has required isolation, in vitro labeling, and reintroduction of the protein back into the cells. We describe a completely biosynthetic approach that avoids these steps. It exploits genetically encodable lanthanide-binding tags (LBT) to form self-assembling Gd(III) metal-based spin labels and enables direct in-cell measurements. This approach is demonstrated using a pair of LBTs encoded one at each end of a 3-helix bundle expressed in E. coli grown on Gd(III) -supplemented medium. DEER measurements directly on these cells produced readily detectable time traces from which the distance between the Gd(III) labels could be determined. This work is the first to use biosynthetically produced self-assembling metal-containing spin labels for non-disruptive in-cell structural measurements. PMID:27496179

  14. Using Genetically Encodable Self-Assembling Gd(III) Spin Labels To Make In-Cell Nanometric Distance Measurements.

    PubMed

    Mascali, Florencia C; Ching, H Y Vincent; Rasia, Rodolfo M; Un, Sun; Tabares, Leandro C

    2016-09-01

    Double electron-electron resonance (DEER) can be used to study the structure of a protein in its native cellular environment. Until now, this has required isolation, in vitro labeling, and reintroduction of the protein back into the cells. We describe a completely biosynthetic approach that avoids these steps. It exploits genetically encodable lanthanide-binding tags (LBT) to form self-assembling Gd(III) metal-based spin labels and enables direct in-cell measurements. This approach is demonstrated using a pair of LBTs encoded one at each end of a 3-helix bundle expressed in E. coli grown on Gd(III) -supplemented medium. DEER measurements directly on these cells produced readily detectable time traces from which the distance between the Gd(III) labels could be determined. This work is the first to use biosynthetically produced self-assembling metal-containing spin labels for non-disruptive in-cell structural measurements.

  15. Biological characteristics of adipose tissue-derived stem cells labeled with amine-surface-modified superparamagnetic iron oxide nanoparticles.

    PubMed

    Wang, Nan; Zhao, Jing-Yuan; Guan, Xin; Dong, Yue; Liu, Yang; Zhou, Xiang; Wu, Ren'an; Du, Yue; Zhao, Liang; Zou, Wei; Han, Chao; Song, Lin; Sun, Bo; Liu, Yan; Liu, Jing

    2015-08-01

    Cell labeling and tracking are becoming increasingly important areas within the field of stem cell transplantation. The ability to track the migration and distribution of implanted cells is critical to understanding the beneficial effects and mechanisms of stem cell therapy. The present study investigated the effects of amine-surface-modified superparamagnetic iron oxide (SPIO) nanoparticles on the biological properties of human adipose tissue-derived stem cells (hADSCs). Monodisperse hydrophobic magnetite (Fe3 O4 ) nanoparticles were prepared using silicon and surface-modified with amine coating. Cell viability, proliferation, differentiation potential, and surface marker expression were evaluated. The magnetic particles (10-18 nm) displayed high labeling efficiency and stability in hADSCs. SPIO-labeled cells produced a hypointense signal and were effectively visualized by MRI for up to 21 days. The results of MTT proliferation assays and flow cytometry analysis demonstrated that SPIOs were biocompatible, viz. the labeling process did not cause cell death or apoptosis and had no side effects on cell proliferation. In vivo experiments showed that the magnetic particles did not affect liver and kidney function. The successful and stable labeling of hADSCs combined with efficient magnetic tropism demonstrates that SPIOs are promising candidates for hADSC tracking in hADSC-based cell therapy applications.

  16. In Vivo Transfer of Intracellular Labels from Locally Implanted Bone Marrow Stromal Cells to Resident Tissue Macrophages

    PubMed Central

    Pawelczyk, Edyta; Jordan, Elaine K.; Balakumaran, Arun; Chaudhry, Aneeka; Gormley, Nicole; Smith, Melissa; Lewis, Bobbi K.; Childs, Richard; Robey, Pamela G.; Frank, Joseph A.

    2009-01-01

    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. PMID:19696933

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

    PubMed

    Ni, Zhongqiu; Zhou, Lanxia; Li, Xu; Zhang, Jing; Dong, Shouliang

    2015-01-01

    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.

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

    PubMed Central

    Ni, Zhongqiu; Zhou, Lanxia; Li, Xu; Zhang, Jing; Dong, Shouliang

    2015-01-01

    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. PMID:26536589

  19. Dynamic analysis of CO₂ labeling and cell respiration using membrane-inlet mass spectrometry.

    PubMed

    Yang, Tae Hoon

    2014-01-01

    Here, we introduce a mass spectrometry-based analytical method and relevant technical details for dynamic cell respiration and CO2 labeling analysis. Such measurements can be utilized as additional information and constraints for model-based (13)C metabolic flux analysis. Dissolved dynamics of oxygen consumption and CO2 mass isotopomer evolution from (13)C-labeled tracer substrates through different cellular processes can be precisely measured on-line using a miniaturized reactor system equipped with a membrane-inlet mass spectrometer. The corresponding specific rates of physiologically relevant gases and CO2 mass isotopomers can be quantified within a short-term range based on the liquid-phase dynamics of dissolved fermentation gases.

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

    NASA Astrophysics Data System (ADS)

    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.

  1. Responses of BrdU label-retaining dental pulp cells to allogenic tooth transplantation into mouse maxilla.

    PubMed

    Mutoh, Noriko; Nakatomi, Mitsushiro; Ida-Yonemochi, Hiroko; Nakagawa, Eizo; Tani-Ishii, Nobuyuki; Ohshima, Hayato

    2011-12-01

    Recently, we demonstrated that a pulse of BrdU given to prenatal animals reveals the existence of slow-cycling long-term label-retaining cells (LRCs), putative adult stem or progenitor cells, which reside in the dental pulp. This study aims to clarify responses of LRCs to allogenic tooth transplantation into mouse maxilla using prenatal BrdU-labeling, in situ hybridization for osteopontin and periostin, and immunohistochemistry for BrdU, nestin, and osteopontin. The upper-right first molars were allografted in the original socket between BrdU-labeled and non-labeled mice or between GFP transgenic and wild-type mice. Tooth transplantation caused degeneration of the odontoblast layer, resulting in the disappearance of nestin-positive reactions in the dental pulp. On postoperative days 5-7, tertiary dentin formation commenced next to the preexisting dentin where nestin-positive odontoblast-like cells were arranged in the successful cases. In BrdU-labeled transplanted teeth, dense LRCs were maintained in the center of the dental pulp beneath the odontoblast-like cells including LRCs, whereas LRCs disappeared in the area surrounding the bone-like tissue. In contrast, LRCs were not recognized in the pulp chamber of non-labeled transplants through the experimental period. Tooth transplantation using GFP mice demonstrated that the donor cells constituted the dental pulp of the transplant except for endothelial cells and some migrated cells, and the periodontal tissue was replaced by host-derived cells except for epithelial cell rests of Malassez. These results suggest that the maintenance of BrdU label-retaining dental pulp cells play a role in the regeneration of odontoblast-like cells in the process of pulpal healing following tooth transplantation. PMID:21986880

  2. Near-infrared emitting fluorescent nanocrystals-labeled natural killer cells as a platform technology for the optical imaging of immunotherapeutic cells-based cancer therapy

    NASA Astrophysics Data System (ADS)

    Taik Lim, Yong; Cho, Mi Young; Noh, Young-Woock; Chung, Jin Woong; Chung, Bong Hyun

    2009-11-01

    This study describes the development of near-infrared optical imaging technology for the monitoring of immunotherapeutic cell-based cancer therapy using natural killer (NK) cells labeled with fluorescent nanocrystals. Although NK cell-based immunotherapeutic strategies have drawn interest as potent preclinical or clinical methods of cancer therapy, there are few reports documenting the molecular imaging of NK cell-based cancer therapy, primarily due to the difficulty of labeling of NK cells with imaging probes. Human natural killer cells (NK92MI) were labeled with anti-human CD56 antibody-coated quantum dots (QD705) for fluorescence imaging. FACS analysis showed that the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 have no effect on the cell viability. The effect of anti-human CD56 antibody-coated QD705 labeling on the NK92MI cell function was investigated by measuring interferon gamma (IFN- γ) production and cytolytic activity. Finally, the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 showed a therapeutic effect similar to that of unlabeled NK92MI cells. Images of intratumorally injected NK92MI cells labeled with anti-human CD56 antibody-coated could be acquired using near-infrared optical imaging both in vivo and in vitro. This result demonstrates that the immunotherapeutic cells labeled with fluorescent nanocrystals can be a versatile platform for the effective tracking of injected therapeutic cells using optical imaging technology, which is very important in cell-based cancer therapies.

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

    PubMed

    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.

  4. Optical painting and fluorescence activated sorting of single adherent cells labelled with photoswitchable Pdots

    PubMed Central

    Kuo, Chun-Ting; Thompson, Alison M.; Gallina, Maria Elena; Ye, Fangmao; Johnson, Eleanor S.; Sun, Wei; Zhao, Mengxia; Yu, Jiangbo; Wu, I-Che; Fujimoto, Bryant; DuFort, Christopher C.; Carlson, Markus A.; Hingorani, Sunil R.; Paguirigan, Amy L.; Radich, Jerald P.; Chiu, Daniel T.

    2016-01-01

    The efficient selection and isolation of individual cells of interest from a mixed population is desired in many biomedical and clinical applications. Here we show the concept of using photoswitchable semiconducting polymer dots (Pdots) as an optical ‘painting' tool, which enables the selection of certain adherent cells based on their fluorescence, and their spatial and morphological features, under a microscope. We first develop a Pdot that can switch between the bright (ON) and dark (OFF) states reversibly with a 150-fold contrast ratio on irradiation with ultraviolet or red light. With a focused 633-nm laser beam that acts as a ‘paintbrush' and the photoswitchable Pdots as the ‘paint', we select and ‘paint' individual Pdot-labelled adherent cells by turning on their fluorescence, then proceed to sort and recover the optically marked cells (with 90% recovery and near 100% purity), followed by genetic analysis. PMID:27118210

  5. Label-free assessment of replicative senescence in mesenchymal stem cells by Raman microspectroscopy

    PubMed Central

    Bai, Hua; Li, Haiyu; Han, Zhibo; Zhang, Cheng; Zhao, Junfa; Miao, Changyun; Yan, Shulin; Mao, Aibin; Zhao, Hui; Han, Zhongchao

    2015-01-01

    Here, Raman microspectroscopy was employed to assess replicative senescence of mesenchymal stem cells (MSC). A regular spectral change related to the cell senescence was found in the ratio of two peaks at 1157 cm−1 and 1174 cm−1, which are assigned to C-C, C-N stretching vibrations in proteins and C-H bending vibrations in tyrosine and phenylalanine, respectively. With the cell aging, the ratio I1157 / I1174 exhibited a monotonic decline and showed small standard deviations, so that it can statistically distinguish between cells having slight changes in terms of aging. We propose that I1157 / I1174 can act as a characteristic spectral signature for label-free assessment of MSC senescence. PMID:26601012

  6. Bone marrow long label-retaining cells reside in the sinusoidal hypoxic niche

    SciTech Connect

    Kubota, Yoshiaki; Takubo, Keiyo; Suda, Toshio

    2008-02-08

    In response to changing signals, quiescent hematopoietic stem cells (HSCs) can be induced to an activated cycling state and provide multi-lineage hematopoietic cells to the whole body via blood vessels. However, the precise localization of quiescent HSCs in bone marrow microenvironment is not fully characterized. Here, we performed whole-mount immunostaining of bone marrow and found that BrdU label-retaining cells (LRCs) definitively reside in the sinusoidal hypoxic zone distant from the 'vascular niche'. Although LRCs expressed very low level of a well-known HSC marker, c-kit in normal circumstances, myeloablation by 5-FU treatment caused LRCs to abundantly express c-kit and proliferate actively. These results demonstrate that bone marrow LRCs reside in the sinusoidal hypoxic niche, and function as a regenerative cell pool of HSCs.

  7. Labeling Live Cells by Copper-Catalyzed Alkyne-Azide Click Chemistry

    PubMed Central

    Hong, Vu; Steinmetz, Nicole F.; Manchester, Marianne

    2010-01-01

    The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, optimized for biological molecules in aqueous buffers, has been shown to rapidly label mammalian cells in culture with no loss in cell viability. Metabolic uptake and display of the azide derivative of N-acetylmannosamine developed by Bertozzi, followed by CuAAC ligation using sodium ascorbate and the ligand tris(hydroxypropyltriazolyl)methylamine (THPTA), gave rise to abundant covalent attachment of dye-alkyne reactants. THPTA serves both to accelerate the CuAAC reaction and to protect the cells from damage by oxidative agents produced by the Cu-catalyzed reduction of oxygen by ascorbate, which is required to maintain the metal in the active +1 oxidation state. This procedure extends the application of this fastest of azide-based bioorthogonal reactions to the exterior of living cells. PMID:20886827

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

    NASA Astrophysics Data System (ADS)

    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.

  9. Efficacy of astatine-211-labeled monoclonal antibody in treatment of murine T-cell lymphoma

    SciTech Connect

    Harrison, A.; Royle, L.

    1987-01-01

    The short-lived isotope /sup 211/At (half-life, 7.2 hr), an alpha particle-emitting halogen, has been attached to a monoclonal antibody (anti-thy 1.1, IgG1, OX7) and used in mice in the treatment of a thy 1.1 T-cell lymphoma (A120). Forty-eight hours after receiving an iv injection of 10(3) or 10(5) A120 cells, mice were treated with phosphate-buffered saline, /sup 211/At-, antibody alone, or /sup 211/At conjugated to OX7. Treatment with the /sup 211/At-labeled OX7 conjugate increased the median survival time of mice and probably cured (survival at 200 days) 6 of the 15 mice given 10(5) cells and 21 of the 27 mice given 10(3) cells.

  10. Efficacy of astatine-211-labeled monoclonal antibody in treatment of murine T-cell lymphoma.

    PubMed

    Harrison, A; Royle, L

    1987-01-01

    The short-lived isotope 211At (half-life, 7.2 hr), an alpha particle-emitting halogen, has been attached to a monoclonal antibody (anti-thy 1.1, IgG1, OX7) and used in mice in the treatment of a thy 1.1 T-cell lymphoma (A120). Forty-eight hours after receiving an iv injection of 10(3) or 10(5) A120 cells, mice were treated with phosphate-buffered saline, 211At-, antibody alone, or 211At conjugated to OX7. Treatment with the 211At-labeled OX7 conjugate increased the median survival time of mice and probably "cured" (survival at 200 days) 6 of the 15 mice given 10(5) cells and 21 of the 27 mice given 10(3) cells.

  11. Label-free biochemical characterization of bovine sperm cells using Raman microscopy

    NASA Astrophysics Data System (ADS)

    De Luca, A. C.; Manago, S.; Ferrara, M. A.; Sirleto, L.; Puglisi, R.; Balduzzi, D.; Galli, A.; Rendina, I.; Ferraro, P.; Coppola, G.

    2013-04-01

    Raman spectroscopy is a label-free and non-invasive method that measures the inelastic scattered light from a sample giving insight into the vibration eigenmodes of the excited molecules. For these reasons, Raman spectroscopy has been used as a powerful tool to investigate different biological tissues and living cells. In this paper, we present a Raman spectroscopy-based method for sensitive biochemical characterization of bovine sperm cells. Importantly, by analysing separate Raman spectra from the nucleus, acrosomale vesicle and tail of single sperm cells, we are able to identify characteristic Raman features associated with DNA, protein and lipid molecular vibrations for discriminating among different locations inside the cell with sub-micrometric resolution (˜0.3 μm). We demonstrate that our Raman spectroscopy facilitates spectral assignment and increases detection sensitivity, opening the way for novel bio-imaging platforms.

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

    PubMed Central

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

    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 fluorescent magnetic nanoparticles (FMNPs) for targeted imaging and synergistic therapy of gastric cancer cells in 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. 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. Results 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. Conclusion FMNP-labeled human iPS cells exhibit considerable potential in applications such as targeted dual-mode imaging and synergistic therapy for early gastric cancer. PMID:26487961

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

    PubMed Central

    Ren, Jianwei; Yao, Peng; Wang, Xiaowei; Wang, Zhe; Zhang, Qunye

    2016-01-01

    Background 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. Methods and Results 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. Conclusions 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. PMID

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

    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

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

    PubMed Central

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

    2016-01-01

    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. PMID:27225821

  17. Genetically engineered, biarsenically labeled influenza virus allows visualization of viral NS1 protein in living cells.

    PubMed

    Li, Yang; Lu, Xinya; Li, Junwei; Bérubé, Nathalie; Giest, Kerri-Lane; Liu, Qiang; Anderson, Deborah H; Zhou, Yan

    2010-07-01

    Real-time fluorescence imaging of viral proteins in living cells provides a valuable means to study virus-host interactions. The challenge of generating replication-competent fluorescent influenza A virus is that the segmented genome does not allow fusion of a fluorescent protein gene to any viral gene. Here, we introduced the tetracysteine (TC) biarsenical labeling system into influenza virus in order to fluorescently label viral protein in the virus life cycle. We generated infectious influenza A viruses bearing a small TC tag (CCPGCC) in the loop/linker regions of the NS1 proteins. In the background of A/Puerto Rico/8/34 (H1N1) (PR8) virus, the TC tag can be inserted into NS1 after amino acid 52 (AA52) (PR8-410), AA79 (PR8-412), or AA102 (PR8-413) or the TC tag can be inserted and replace amino acids 79 to 84 (AA79-84) (PR8-411). Although PR8-410, PR8-411, and PR8-412 viruses are attenuated than the wild-type (WT) virus to some extent in multiple-cycle infection, their growth potential is similar to that of the WT virus during a single cycle of infection, and their NS1 subcellular localization and viral protein synthesis rate are quite similar to those of the WT virus. Furthermore, labeling with membrane-permeable biarsenical dye resulted in fluorescent NS1 protein in the context of virus infection. We could exploit this strategy on NS1 protein of A/Texas/36/91 (H1N1) (Tx91) by successfully rescuing a TC-tagged virus, Tx91-445, which carries the TC tag replacement of AA79-84. The infectivity of Tx91-445 virus was similar to that of WT Tx91 during multiple cycles of replication and a single cycle of replication. The NS1 protein derived from Tx91-445 can be fluorescently labeled in living cells. Finally, with biarsenical labeling, the engineered replication-competent virus allowed us to visualize NS1 protein nuclear import in virus-infected cells in real time.

  18. Noninvasive and label-free detection of circulating melanoma cells by in vivo photoacoustic flow cytometry

    NASA Astrophysics Data System (ADS)

    Yang, Ping; Liu, Rongrong; Niu, Zhenyu; Suo, Yuanzhen; He, Hao; Wei, Xunbin

    2015-03-01

    Melanoma is a malignant tumor of melanocytes. Circulating melanoma cell has 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). PAFC 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. In our research, we developed in vitro experiments to prove the ability of PAFC system of detecting PA signals from melanoma cells. For in vivo experiments, we constructed a model of melanoma tumor bearing mice by inoculating highly metastatic murine melanoma cancer cells B16F10 with subcutaneous injection. PA signals were detected in the blood vessels of mouse ears in vivo. By counting circulating melanoma cells termly, we obtained 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. Our PAFC system is an efficient tool to monitor melanoma metastases, cancer recurrence and therapeutic efficacy.

  19. High Throughput Label Free Measurement of Cancer Cell Adhesion Kinetics Under Hemodynamic Flow

    PubMed Central

    Spencer, Adrianne; Baker, Aaron B.

    2016-01-01

    The kinetics of receptor-mediated cell adhesion to extracellular matrix and adherent cell monolayers plays a key role in many physiological and pathological processes including cancer metastasis. Within this process the presence of fluidic shear forces is a key regulator of binding equilibrium and kinetics of cell adhesion. Current techniques to examine the kinetics of cell adhesion are either performed in the absence of flow or are low throughput, limiting their application to pharmacological compound screening or the high throughput investigation of biological mechanisms. We developed a high throughput flow device that applies flow in a multi-well format and interfaced this system with electric cell-substrate impedance sensing (ECIS) system to allow label free detection of cell adhesion. We demonstrate that this combined system is capable of making real time measurements of cancer cell adhesion to extracellular matrix and immobilized platelets. In addition, we examined the dependence of the kinetics of binding of cancer cells on the level of shear stress and in the presence of small molecule inhibitors to adhesion-related pathways. This versatile system is broadly adaptable to the high throughput study of cell adhesion kinetics for many applications including drug screening and the investigation of the mechanisms of cancer metastasis. PMID:26816215

  20. Cryopreservation of embryonic stem cell-derived multicellular neural aggregates labeled with micron-sized particles of iron oxide for magnetic resonance imaging.

    PubMed

    Yan, Yuanwei; Sart, Sébastien; Calixto Bejarano, Fabian; Muroski, Megan E; Strouse, Geoffrey F; Grant, Samuel C; Li, Yan

    2015-01-01

    Magnetic resonance imaging (MRI) provides an effective approach to track labeled pluripotent stem cell (PSC)-derived neural progenitor cells (NPCs) for neurological disorder treatments after cell labeling with a contrast agent, such as an iron oxide derivative. Cryopreservation of pre-labeled neural cells, especially in three-dimensional (3D) structure, can provide a uniform cell population and preserve the stem cell niche for the subsequent applications. In this study, the effects of cryopreservation on PSC-derived multicellular NPC aggregates labeled with micron-sized particles of iron oxide (MPIO) were investigated. These NPC aggregates were labeled prior to cryopreservation because labeling thawed cells can be limited by inefficient intracellular uptake, variations in labeling efficiency, and increased culture time before use, minimizing their translation to clinical settings. The results indicated that intracellular MPIO incorporation was retained after cryopreservation (70-80% labeling efficiency), and MPIO labeling had little adverse effects on cell recovery, proliferation, cytotoxicity and neural lineage commitment post-cryopreservation. MRI analysis showed comparable detectability for the MPIO-labeled cells before and after cryopreservation indicated by T2 and T2* relaxation rates. Cryopreserving MPIO-labeled 3D multicellular NPC aggregates can be applied in in vivo cell tracking studies and lead to more rapid translation from preservation to clinical implementation. PMID:25905549

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

    NASA Astrophysics Data System (ADS)

    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.

  2. Perylene-labeled silica nanoparticles: synthesis and characterization of three novel silica nanoparticle species for live-cell imaging.

    PubMed

    Blechinger, Julia; Herrmann, Rudolf; Kiener, Daniel; García-García, F Javier; Scheu, Christina; Reller, Armin; Bräuchle, Christoph

    2010-11-01

    The increasing exposure of humans to nanoscaled particles requires well-defined systems that enable the investigation of the toxicity of nanoparticles on the cellular level. To facilitate this, surface-labeled silica nanoparticles, nanoparticles with a labeled core and a silica shell, and a labeled nanoparticle network-all designed for live-cell imaging-are synthesized. The nanoparticles are functionalized with perylene derivatives. For this purpose, two different perylene species containing one or two reactive silica functionalities are prepared. The nanoparticles are studied by transmission electron microscopy, widefield and confocal fluorescence microscopy, as well as by fluorescence spectroscopy in combination with fluorescence anisotropy, in order to characterize the size and morphology of the nanoparticles and to prove the success and homogeneity of the labeling. Using spinning-disc confocal measurements, silica nanoparticles are demonstrated to be taken up by HeLa cells, and they are clearly detectable inside the cytoplasm of the cells.

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

    PubMed

    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

  4. When cells divide: Label-free multimodal spectral imaging for exploratory molecular investigation of living cells during cytokinesis

    NASA Astrophysics Data System (ADS)

    Hsu, Jen-Fang; Hsieh, Pei-Ying; Hsu, Hsin-Yun; Shigeto, Shinsuke

    2015-12-01

    In vivo, molecular-level investigation of cytokinesis, the climax of the cell cycle, not only deepens our understanding of how life continues, but it will also open up new possibilities of diagnosis/prognosis of cancer cells. Although fluorescence-based methods have been widely employed to address this challenge, they require a fluorophore to be designed for a specific known biomolecule and introduced into the cell. Here, we present a label-free spectral imaging approach based on multivariate curve resolution analysis of Raman hyperspectral data that enables exploratory untargeted studies of mammalian cell cytokinesis. We derived intrinsic vibrational spectra and intracellular distributions of major biomolecular components (lipids and proteins) in dividing and nondividing human colon cancer cells. In addition, we discovered an unusual autofluorescent lipid component that appears predominantly in the vicinity of the cleavage furrow during cytokinesis. This autofluorescence signal could be utilized as an endogenous probe for monitoring and visualizing cytokinesis in vivo.

  5. When cells divide: Label-free multimodal spectral imaging for exploratory molecular investigation of living cells during cytokinesis

    PubMed Central

    Hsu, Jen-Fang; Hsieh, Pei-Ying; Hsu, Hsin-Yun; Shigeto, Shinsuke

    2015-01-01

    In vivo, molecular-level investigation of cytokinesis, the climax of the cell cycle, not only deepens our understanding of how life continues, but it will also open up new possibilities of diagnosis/prognosis of cancer cells. Although fluorescence-based methods have been widely employed to address this challenge, they require a fluorophore to be designed for a specific known biomolecule and introduced into the cell. Here, we present a label-free spectral imaging approach based on multivariate curve resolution analysis of Raman hyperspectral data that enables exploratory untargeted studies of mammalian cell cytokinesis. We derived intrinsic vibrational spectra and intracellular distributions of major biomolecular components (lipids and proteins) in dividing and nondividing human colon cancer cells. In addition, we discovered an unusual autofluorescent lipid component that appears predominantly in the vicinity of the cleavage furrow during cytokinesis. This autofluorescence signal could be utilized as an endogenous probe for monitoring and visualizing cytokinesis in vivo. PMID:26632877

  6. Rapid synthesis of PEGylated ultrasmall gadolinium oxide nanoparticles for cell labeling and tracking with MRI.

    PubMed

    Faucher, Luc; Tremblay, Mélanie; Lagueux, Jean; Gossuin, Yves; Fortin, Marc-André

    2012-09-26

    Ultrasmall paramagnetic Gd(2)O(3) nanoparticles have been developed as contrast agents for molecular and cellular preclinical MRI procedures. These small particles (mean diameter <5 nm) have the highest Gd density of all paramagnetic contrast agents. They generate strong positive contrast enhancement in T(1)-weighted MRI. Signal enhancement is modulated by the interactions of water molecules with Gd, and very small particles provide the optimal surface-to-volume ratios necessary to reach high relaxivities. Conventional Gd(2)O(3) nanocrystal synthesis techniques, and subsequent polyethylene glycol (PEG) grafting procedures are usually time-consuming and recovery losses are also limitative. The present study reports on a new, fast, and efficient one-pot Gd(2)O(3) synthesis technique that provides PEGylated nanoparticles of very small size (mean diameter = 1.3 nm). Readily coated with PEG, the particles are colloidally stable in aqueous media and provide high longitudial relaxivities and small r(2)/r(1) ratios (r(1) = 14.2 mM(-1) s(-1) at 60 MHz; r(2)/r(1) = 1.20), ideal for T(1)-weighted MRI. In this study, F98 brain cancer cells (glioblastoma multiforme) were labeled with the contrast agent and implanted in vivo (mice brains). The labeled cells appeared positively contrasted at least 48 h after implantation. Each one of the implanted animals developed a brain tumor. The performance of PEG-Gd(2)O(3) was also compared with that of commercially available iron oxide nanoparticles. This study demonstrated that ultrasmall PEG-Gd(2)O(3) nanoparticles provide strong positive contrast enhancement in T(1)-weighted imaging, and allow the visualization of labeled cells implanted in vivo.

  7. Cellular processing of copper-67-labeled monoclonal antibody chCE7 by human neuroblastoma cells.

    PubMed

    Novak-Hofer, I; Amstutz, H P; Mäcke, H R; Schwarzbach, R; Zimmermann, K; Morgenthaler, J J; Schubiger, P A

    1995-01-01

    Monoclonal antibody chCE7, an internalizing neuroblastoma-specific chimeric antibody, was derivatized with the macrocyclic amine ligand 4-[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl] benzoic acid tetrahydrochloride and labeled with the potential therapeutic nuclide 67Cu. Using pulse labeling and an acid elution endocytosis assay, 67Cu-chCE7 was found to be internalized into human neuroblastoma (SKN-AS) cells at a similar rate and to a similar extent as 125I-labeled chCE7. Uptake of 67Cu-chCE7 and 125I-chCE7 into the acid stable (intracellular) pool proceeded with similar kinetics during the first 2 h of internalization. However, in contrast to 125I-chCE7-loaded cells, at later times intracellular radioactivity kept increasing in the case of 67Cu-chCE7-loaded cells. It was shown that this effect is due to the intracellular accumulation of a low M(r) degradation product consisting of the 67Cu-4[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl] benzoic acid complex, possibly with a short peptide attached to it. Degradation of both 125I-chCE7 and 67Cu-chCE7 was inhibited by chloroquine, indicating endosomal or lysosomal degradation, and a 43,000 M(r) fragment was found to be the major high M(r) degradation product in both cases. Although at times between 4 and 6 h of internalization intracellular breakdown of 67Cu-chCE7 was found to proceed more slowly, the major difference between the two immunoconjugates resides in the prolonged cellular retention of the 67Cu-chCE7 metabolite.

  8. Cellular processing of copper-67-labeled monoclonal antibody chCE7 by human neuroblastoma cells.

    PubMed

    Novak-Hofer, I; Amstutz, H P; Mäcke, H R; Schwarzbach, R; Zimmermann, K; Morgenthaler, J J; Schubiger, P A

    1995-01-01

    Monoclonal antibody chCE7, an internalizing neuroblastoma-specific chimeric antibody, was derivatized with the macrocyclic amine ligand 4-[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl] benzoic acid tetrahydrochloride and labeled with the potential therapeutic nuclide 67Cu. Using pulse labeling and an acid elution endocytosis assay, 67Cu-chCE7 was found to be internalized into human neuroblastoma (SKN-AS) cells at a similar rate and to a similar extent as 125I-labeled chCE7. Uptake of 67Cu-chCE7 and 125I-chCE7 into the acid stable (intracellular) pool proceeded with similar kinetics during the first 2 h of internalization. However, in contrast to 125I-chCE7-loaded cells, at later times intracellular radioactivity kept increasing in the case of 67Cu-chCE7-loaded cells. It was shown that this effect is due to the intracellular accumulation of a low M(r) degradation product consisting of the 67Cu-4[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl] benzoic acid complex, possibly with a short peptide attached to it. Degradation of both 125I-chCE7 and 67Cu-chCE7 was inhibited by chloroquine, indicating endosomal or lysosomal degradation, and a 43,000 M(r) fragment was found to be the major high M(r) degradation product in both cases. Although at times between 4 and 6 h of internalization intracellular breakdown of 67Cu-chCE7 was found to proceed more slowly, the major difference between the two immunoconjugates resides in the prolonged cellular retention of the 67Cu-chCE7 metabolite. PMID:7805039

  9. A Recombinant Fungal Lectin for Labeling Truncated Glycans on Human Cancer Cells

    PubMed Central

    Hurbin, Amandine; Boos, Irene; Unverzagt, Carlo; Bouras, Mourad; Lantuejoul, Sylvie; Coll, Jean-Luc; Varrot, Annabelle; Le Pendu, Jacques; Busser, Benoit; Imberty, Anne

    2015-01-01

    Cell surface glycoconjugates present alterations of their structures in chronic diseases and distinct oligosaccharide epitopes have been associated with cancer. Among them, truncated glycans present terminal non-reducing β-N-acetylglucosamine (GlcNAc) residues that are rare on healthy tissues. Lectins from unconventional sources such as fungi or algi provide novel markers that bind specifically to such epitopes, but their availability may be challenging. A GlcNAc-binding lectin from the fruiting body of the fungus Psathyrella velutina (PVL) has been produced in good yield in bacterial culture. A strong specificity for terminal GlcNAc residues was evidenced by glycan array. Affinity values obtained by microcalorimetry and surface plasmon resonance demonstrated a micromolar affinity for GlcNAcβ1-3Gal epitopes and for biantennary N-glycans with GlcNAcβ1-2Man capped branches. Crystal structure of PVL complexed with GlcNAcβ1-3Gal established the structural basis of the specificity. Labeling of several types of cancer cells and use of inhibitors of glycan metabolism indicated that rPVL binds to terminal GlcNAc but also to sialic acid (Neu5Ac). Analysis of glycosyltransferase expression confirmed the higher amount of GlcNAc present on cancer cells. rPVL binding is specific to cancer tissue and weak or no labeling is observed for healthy ones, except for stomach glands that present unique αGlcNAc-presenting mucins. In lung, breast and colon carcinomas, a clear delineation could be observed between cancer regions and surrounding healthy tissues. PVL is therefore a useful tool for labeling agalacto-glycans in cancer or other diseases. PMID:26042789

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

    PubMed

    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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26324940

  12. Microfluidic devices for label-free separation of cells through transient interaction with asymmetric receptor patterns

    NASA Astrophysics Data System (ADS)

    Bose, S.; Singh, R.; Hollatz, M. H.; Lee, C.-H.; Karp, J.; Karnik, R.

    2012-02-01

    Cell sorting serves an important role in clinical diagnosis and biological research. Most of the existing microscale sorting techniques are either non-specific to antigen type or rely on capturing cells making sample recovery difficult. We demonstrate a simple; yet effective technique for isolating cells in an antigen specific manner by using transient interactions of the cell surface antigens with asymmetric receptor patterned surface. Using microfluidic devices incorporating P-selectin patterns we demonstrate separation of HL60 cells from K562 cells. We achieved a sorting purity above 90% and efficiency greater than 85% with this system. We also present a mathematical model incorporating flow mediated and adhesion mediated transport of cells in the microchannel that can be used to predict the performance of these devices. Lastly, we demonstrate the clinical significance of the method by demonstrating single step separation of neutrophils from whole blood. When whole blood is introduced in the device, the granulocyte population gets separated exclusively yielding neutrophils of high purity (<10% RBC contamination). To our knowledge, this is the first ever demonstration of continuous label free sorting of neutrophils from whole blood. We believe this technology will be useful in developing point-of-care diagnostic devices and also for a host of cell sorting applications.

  13. Mechanics of swimming of multi-body bacterial swarmers using non-labeled cell tracking algorithm

    NASA Astrophysics Data System (ADS)

    Phuyal, Kiran; Kim, Min Jun

    2013-01-01

    To better understand the survival strategy of bacterial swarmers and the mechanical advantages offered by the linear chain (head-tail) attachment of the multiple bacterial bodies in an individual swarmer cell at low Reynolds number, a non-labeled cell tracking algorithm was used to quantify the mechanics of multi-body flagellated bacteria, Serratia marcescens, swimming in a motility buffer that originally exhibited the swarming motility. Swarming is a type of bacterial motility that is characterized by the collective coordinated motion of differentiated swarmer cells on a two-dimensional surface such as agar. In this study, the bacterial swarmers with multiple cell bodies (2, 3, and 4) were extracted from the swarm plate, and then tracked individually after resuspending in the motility medium. Their motion was investigated and compared with individual undifferentiated swimming bacterial cells. The swarmers when released into the motility buffer swam actively without tumbles. Their speeds, orientations, and the diffusive properties were studied by tracking the individual cell trajectories over a short distance in two-dimensional field when the cells are swimming at a constant depth in a bulk aqueous environment. At short time scales, the ballistic trajectory was dominant for both multi-body swarmers and undifferentiated cells.

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

    PubMed

    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

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

    PubMed Central

    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

  16. Phase-sensitive flow cytometry: New technology for analyzing biochemical, functional, and structural features in fluorochrome- labeled cells/particles

    SciTech Connect

    Steinkamp, J.A.

    1993-12-01

    Flow cytometry (FCM) instruments rapidly measure biochemical, functional, and cytological properties of individual cells and macromolecular components, e.g., chromosomes, for clinical diagnostic medicine and biomedical and envirorunental research applications. These measurements are based on labeling cells with multiple fluorochromes for correlated analysis of macromolecules, such as DNA RNA, protein, and cell-surface receptors. This report describes the development of a phase-sensitive flow cytometer that provides unique capabilities for making laser-excited, phase-resolved measurements on fluorochrome-labelled cells and particles.

  17. Fluorescence polarization of DPH-labeled cells adsorbing viruses and its diagnostic potential.

    PubMed

    Levanon, A; Inbar, M; Kohn, A

    1979-01-01

    Mammalian or avian cells were labeled with a fluorescent probe DPH (1,6-diphenyl-1,3,5-hexatriene). Within a few minutes after adsorption of various naked and enveloped viruses, the degree of fluorescence polarization (P) of the DPH embedded in the adsorbing cells as measured at 37 degrees C, was reduced, a finding indicating a decrease in the microviscosity of the lipids in the cell membrane. This change of fluidity was proportional to the concentration of the adsorbing virus and could be abolished or inhibited by homologous specific antiviral sera, but not by heterologous sera. Potential use of fluorescence polarization tests is described for titration of virus concentration, as well as for serological identification of a virus.

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

    PubMed

    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.

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

    PubMed Central

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

    2008-01-01

    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 CH2-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 CH2 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. PMID:19343073

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

    NASA Astrophysics Data System (ADS)

    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.

  1. Label-Free Imaging and Biochemical Characterization of Bovine Sperm Cells

    PubMed Central

    Ferrara, Maria Antonietta; Di Caprio, Giuseppe; Managò, Stefano; De Angelis, Annalisa; Sirleto, Luigi; Coppola, Giuseppe; De Luca, Anna Chiara

    2015-01-01

    A full label-free morphological and biochemical characterization is desirable to select spermatozoa during preparation for artificial insemination. In order to study these fundamental parameters, we take advantage of two attractive techniques: digital holography (DH) and Raman spectroscopy (RS). DH presents new opportunities for studying morphological aspect of cells and tissues non-invasively, quantitatively and without the need for staining or tagging, while RS is a very specific technique allowing the biochemical analysis of cellular components with a spatial resolution in the sub-micrometer range. In this paper, morphological and biochemical bovine sperm cell alterations were studied using these techniques. In addition, a complementary DH and RS study was performed to identify X- and Y-chromosome-bearing sperm cells. We demonstrate that the two techniques together are a powerful and highly efficient tool elucidating some important criterions for sperm morphological selection and sex-identification, overcoming many of the limitations associated with existing protocols. PMID:25836358

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

    PubMed

    Maltas, Esra; Malkondu, Sait; Uyar, Pembegul; Ozmen, Mustafa

    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 25mg of nanoparticle concentration for Calf thymus DNA. Binding capacity of PBI-TRIS to DNA-SPION was also found as 1.93μM on 25mg 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 561.7nm of excitation wavelengths. Cell image was also compared with a commercial dye, DAPI at 403.7nm of excitation wavelength. Results showed that PBI-TRIS can be used for cell staining.

  3. Technetium labelled red blood cell scintigraphy in the diagnosis of intestinal haemorrhage.

    PubMed Central

    Harvey, M. H.; Neoptolemos, J. P.; Watkin, E. M.; Cosgriff, P.; Barrie, W. W.

    1985-01-01

    99m-Technetium labelled red blood cell scintigraphy was used in the investigation of 15 adult patients with suspected small or large bowel bleeding requiring at least five units of blood (mean 14.3 units) and one neonate with rectal bleeding. Scintigraphy was found to be an accurate method of detecting the site of haemorrhage and was superior to angiography. This technique may be of particular value in patients with profuse colonic haemorrhage when the view at colonoscopy is poor. Images Fig. 1 Fig. 2 Fig. 3 PMID:3872094

  4. Highly Stable trans-Cyclooctene Amino Acids for Live-Cell Labeling.

    PubMed

    Hoffmann, Jan-Erik; Plass, Tilman; Nikić, Ivana; Aramburu, Iker Valle; Koehler, Christine; Gillandt, Hartmut; Lemke, Edward A; Schultz, Carsten

    2015-08-24

    trans-Cyclooctene groups incorporated into proteins via non-canonical amino acids (ncAAs) are emerging as specific handles for bioorthogonal chemistry. Here, we present a highly improved synthetic access to the axially and the equatorially linked trans-cyclooct-2-ene isomers (1 a,b). We further show that the axially connected isomer has a half-life about 10 times higher than the equatorial isomer and reacts with tetrazines much faster, as determined by stopped-flow experiments. The improved properties resulted in different labeling performance of the insulin receptor on the surface of intact cells.

  5. Node-pore sensing enables label-free surface-marker profiling of single cells.

    PubMed

    Balakrishnan, Karthik R; Whang, Jeremy C; Hwang, Richard; Hack, James H; Godley, Lucy A; Sohn, Lydia L

    2015-03-01

    Flow cytometry is a ubiquitous, multiparametric method for characterizing cellular populations. However, this method can grow increasingly complex with the number of proteins that need to be screened simultaneously: spectral emission overlap of fluorophores and the subsequent need for compensation, lengthy sample preparation, and multiple control tests that need to be performed separately must all be considered. These factors lead to increased costs, and consequently, flow cytometry is performed in core facilities with a dedicated technician operating the instrument. Here, we describe a low-cost, label-free microfluidic method that can determine the phenotypic profiles of single cells. Our method employs Node-Pore Sensing to measure the transit times of cells as they interact with a series of different antibodies, each corresponding to a specific cell-surface antigen, that have been functionalized in a single microfluidic channel. We demonstrate the capabilities of our method not only by screening two acute promyelocytic leukemia human cells lines (NB4 and AP-1060) for myeloid antigens, CD13, CD14, CD15, and CD33, simultaneously, but also by distinguishing a mixture of cells of similar size—AP-1060 and NALM-1—based on surface markers CD13 and HLA-DR. Furthermore, we show that our method can screen complex subpopulations in clinical samples: we successfully identified the blast population in primary human bone marrow samples from patients with acute myeloid leukemia and screened these cells for CD13, CD34, and HLA-DR. We show that our label-free method is an affordable, highly sensitive, and user-friendly technology that has the potential to transform cellular screening at the benchside.

  6. Node-pore sensing enables label-free surface-marker profiling of single cells.

    PubMed

    Balakrishnan, Karthik R; Whang, Jeremy C; Hwang, Richard; Hack, James H; Godley, Lucy A; Sohn, Lydia L

    2015-03-01

    Flow cytometry is a ubiquitous, multiparametric method for characterizing cellular populations. However, this method can grow increasingly complex with the number of proteins that need to be screened simultaneously: spectral emission overlap of fluorophores and the subsequent need for compensation, lengthy sample preparation, and multiple control tests that need to be performed separately must all be considered. These factors lead to increased costs, and consequently, flow cytometry is performed in core facilities with a dedicated technician operating the instrument. Here, we describe a low-cost, label-free microfluidic method that can determine the phenotypic profiles of single cells. Our method employs Node-Pore Sensing to measure the transit times of cells as they interact with a series of different antibodies, each corresponding to a specific cell-surface antigen, that have been functionalized in a single microfluidic channel. We demonstrate the capabilities of our method not only by screening two acute promyelocytic leukemia human cells lines (NB4 and AP-1060) for myeloid antigens, CD13, CD14, CD15, and CD33, simultaneously, but also by distinguishing a mixture of cells of similar size—AP-1060 and NALM-1—based on surface markers CD13 and HLA-DR. Furthermore, we show that our method can screen complex subpopulations in clinical samples: we successfully identified the blast population in primary human bone marrow samples from patients with acute myeloid leukemia and screened these cells for CD13, CD34, and HLA-DR. We show that our label-free method is an affordable, highly sensitive, and user-friendly technology that has the potential to transform cellular screening at the benchside. PMID:25625182

  7. Label-Retaining Stromal Cells in Mouse Endometrium Awaken for Expansion and Repair After Parturition

    PubMed Central

    Cao, Mingzhu; Yeung, William S.B.

    2015-01-01

    Human and mouse endometrium undergo dramatic cellular reorganization during pregnancy and postpartum. Somatic stem cells maintain homeostasis of the tissue by providing a cell reservoir for regeneration. We hypothesized that endometrial cells with quiescent properties (stem/progenitor cells) were involved in the regeneration of the endometrial tissue. Given that stem cells divide infrequently, they can retain the DNA synthesis label [bromodeoxyuridine (BrdU)] after a prolonged chase period. In this study, prepubertal mice were pulsed with BrdU and after a 6-week chase a small population of label-retaining stromal cells (LRSC) was located primarily beneath the luminal epithelium, adjacent to blood vessels, and near the endometrial–myometrial junction. Marker analyses suggested that they were of mesenchymal origin expressing CD44+, CD90+, CD140b+, CD146+, and Sca-1+. During pregnancy, nonproliferating LRSC predominately resided at the interimplantation/placental loci of the gestational endometrium. Immediately after parturition, a significant portion of the LRSC underwent proliferation (BrdU+/Ki-67+) and expressed total and active β-catenin. The β-catenin expression in the LRSC was transiently elevated at postpartum day (PPD) 1. The proliferation of LRSC resulted in a significant decline in the proportion of LRSC in the postpartum uterus. The LRSC returned to dormancy at PPD7, and the percentage of LRSC remained stable thereafter until 11 weeks. This study demonstrated that LRSC can respond efficiently to physiological stimuli upon initiation of uterine involution and return to its quiescent state after postpartum repair. PMID:25386902

  8. Node-Pore Sensing Enables Label-Free Surface-Marker Profiling of Single Cells

    PubMed Central

    2015-01-01

    Flow cytometry is a ubiquitous, multiparametric method for characterizing cellular populations. However, this method can grow increasingly complex with the number of proteins that need to be screened simultaneously: spectral emission overlap of fluorophores and the subsequent need for compensation, lengthy sample preparation, and multiple control tests that need to be performed separately must all be considered. These factors lead to increased costs, and consequently, flow cytometry is performed in core facilities with a dedicated technician operating the instrument. Here, we describe a low-cost, label-free microfluidic method that can determine the phenotypic profiles of single cells. Our method employs Node-Pore Sensing to measure the transit times of cells as they interact with a series of different antibodies, each corresponding to a specific cell-surface antigen, that have been functionalized in a single microfluidic channel. We demonstrate the capabilities of our method not only by screening two acute promyelocytic leukemia human cells lines (NB4 and AP-1060) for myeloid antigens, CD13, CD14, CD15, and CD33, simultaneously, but also by distinguishing a mixture of cells of similar size—AP-1060 and NALM-1—based on surface markers CD13 and HLA-DR. Furthermore, we show that our method can screen complex subpopulations in clinical samples: we successfully identified the blast population in primary human bone marrow samples from patients with acute myeloid leukemia and screened these cells for CD13, CD34, and HLA-DR. We show that our label-free method is an affordable, highly sensitive, and user-friendly technology that has the potential to transform cellular screening at the benchside. PMID:25625182

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

    PubMed

    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

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

    PubMed

    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

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  13. Freeze-fracture cytochemistry: replicas of critical point-dried cells and tissues after fracture-label.

    PubMed

    da Silva, P P; Kachar, B; Torrisi, M R; Brown, C; Parkison, C

    1981-07-10

    Applications of the new fracture-labeling techniques for the observation of cytochemical labels on platinum-carbon replicas are described. Frozen cells, embedded in a cross-linked protein matrix, and frozen tissues are fractured with a scalpel under liquid nitrogen, thawed, labeled, dehydrated by the critical point drying method, and replicated. This method allows direct, high-resolution, two-dimensional chemical and immunological characterization of the cellular membranes in situ, as well as detection of sites within cross-fractured cytoplasm and extracellular matrix. PMID:7244630

  14. Multiexcitation Fluorogenic Labeling of Surface, Intracellular, and Total Protein Pools in Living Cells.

    PubMed

    Naganbabu, Matharishwan; Perkins, Lydia A; Wang, Yi; Kurish, Jeffery; Schmidt, Brigitte F; Bruchez, Marcel P

    2016-06-15

    Malachite green (MG) is a fluorogenic dye that shows fluorescence enhancement upon binding to its engineered cognate protein, a fluorogen activating protein (FAP). Energy transfer donors such as cyanine and rhodamine dyes have been conjugated with MG to modify the spectral properties of the fluorescent complexes, where the donor dyes transfer energy through Förster resonance energy transfer to the MG complex resulting in binding-conditional fluorescence emission in the far-red region. In this article, we use a violet-excitable dye as a donor to sensitize the far-red emission of the MG-FAP complex. Two blue emitting fluorescent coumarin dyes were coupled to MG and evaluated for energy transfer to the MG-FAP complex via its secondary excitation band. 6,8-Difluoro-7-hydroxycoumarin-3-carboxylic acid (Pacific blue, PB) showed the most efficient energy transfer and maximum brightness in the far-red region upon violet (405 nm) excitation. These blue-red (BluR) tandem dyes are spectrally varied from other tandem dyes and are able to produce fluorescence images of the MG-FAP complex with a large Stokes shift (>250 nm). These dyes are cell-permeable and are used to label intracellular proteins. Used together with a cell-impermeable hexa-Cy3-MG (HCM) dye that labels extracellular proteins, we are able to visualize extracellular, intracellular, and total pools of cellular protein using one fluorogenic tag that combines with distinct dyes to effect different spectral characteristics. PMID:27159569

  15. Transformation of (sup14)C-Lignin-Labeled Cell Walls of Wheat by Syntrophococcus sucromutans, Eubacterium oxidoreducens, and Neocallimastix frontalis

    PubMed Central

    Bernard-Vailhe, M. A.; Besle, J. M.; Dore, J.

    1995-01-01

    Wheat cell walls, saponified or not, labeled with [U-(sup14)C]phenylalanine or [O-methyl-(sup14)C]sinapate were fermented by Neocallimastix frontalis or Syntrophococcus sucromutans plus Eubacterium oxidoreducens or a mixed culture. Phenolics were less solubilized but more transformed by bacteria than by the fungus, and mineralization was slight. S. sucromutans O-demethylated [O-methyl-(sup14)C]syringyl lignins, yielding labeled acetate. PMID:16534916

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

    NASA Astrophysics Data System (ADS)

    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.

  17. Non-invasive, label-free cell counting and quantitative analysis of adherent cells using digital holography.

    PubMed

    Mölder, A; Sebesta, M; Gustafsson, M; Gisselson, L; Wingren, A Gjörloff; Alm, K

    2008-11-01

    Manual cell counting is time consuming and requires a high degree of skill on behalf of the person performing the count. Here we use a technique that utilizes digital holography, allowing label-free and completely non-invasive cell counting directly in cell culture vessels with adherent viable cells. The images produced can provide both quantitative and qualitative phase information from a single hologram. The recently constructed microscope Holomonitor (Phase Holographic Imaging AB, Lund, Sweden) combines the commonly used phase contrast microscope with digital holography, the latter giving us the possibility of achieving quantitative information on cellular shape, area, confluence and optical thickness. This project aimed at determining the accuracy and repeatability of cell counting measurements using digital holography compared to the conventional manual cell counting method using a haemocytometer. The collected data were also used to determine cell size and cellular optical thickness. The results show that digital holography can be used for non-invasive automatic cell counting as precisely as conventional manual cell counting. PMID:19017223

  18. Whole-cell biosensor for label-free detection of GPCR-mediated drug responses in personal cell lines.

    PubMed

    Hillger, Julia M; Schoop, Jeffison; Boomsma, Dorret I; Slagboom, P Eline; IJzerman, Adriaan P; Heitman, Laura H

    2015-12-15

    Deciphering how genetic variation in drug targets such as G protein-coupled receptors (GPCRs) affects drug response is essential for precision medicine. GPCR signaling is traditionally investigated in artificial cell lines which do not provide sufficient physiological context. Patient-derived cell lines such as lymphoblastoid cell lines (LCLs) could represent the ideal cellular model system. Here we describe a novel label-free, whole-cell biosensor method for characterizing GPCR-mediated drug responses in LCLs. Generally, such biosensor technology is deemed only compatible with adherent cell lines. We optimized and applied the methodology to study cellular adhesion properties as well as GPCR drug responses in LCLs, which are suspension cells. Coating the detector surface with the extracellular matrix protein fibronectin resulted in cell adherence and allowed detection of cellular responses. A prototypical GPCR present on these cells, i.e. the cannabinoid receptor 2 (CB2), was selected for pharmacological characterization. Receptor activation with the agonist JWH133, blockade by antagonist AM630 as well as downstream signaling inhibition by PTX could be monitored sensitively and receptor-specifically. Potencies and effects were comparable between LCLs of two genetically unrelated individuals, providing the proof-of-principle that this biosensor technology can be applied to LCLs, despite their suspension cell nature, in order to serve as an in vitro model system for the evaluation of individual genetic influences on GPCR-mediated drug responses.

  19. Ultra-fast, label-free isolation of circulating tumor cells from blood using spiral microfluidics.

    PubMed

    Warkiani, Majid Ebrahimi; Khoo, Bee Luan; Wu, Lidan; Tay, Andy Kah Ping; Bhagat, Ali Asgar S; Han, Jongyoon; Lim, Chwee Teck

    2016-01-01

    Circulating tumor cells (CTCs) are rare cancer cells that are shed from primary or metastatic tumors into the peripheral blood circulation. Phenotypic and genetic characterization of these rare cells can provide important information to guide cancer staging and treatment, and thus further research into their characteristics and properties is an area of considerable interest. In this protocol, we describe detailed procedures for the production and use of a label-free spiral microfluidic device to allow size-based isolation of viable CTCs using hydrodynamic forces that are present in curvilinear microchannels. This spiral system enables us to achieve ≥ 85% recovery of spiked cells across multiple cancer cell lines and 99.99% depletion of white blood cells in whole blood. The described spiral microfluidic devices can be produced at an extremely low cost using standard microfabrication and soft lithography techniques (2-3 d), and they can be operated using two syringe pumps for lysed blood samples (7.5 ml in 12.5 min for a three-layered multiplexed chip). The fast processing time and the ability to collect CTCs from a large patient blood volume allows this technique to be used experimentally in a broad range of potential genomic and transcriptomic applications. PMID:26678083

  20. Highly permeable silicon membranes for shear free chemotaxis and rapid cell labeling.

    PubMed

    Chung, Henry H; Chan, Charles K; Khire, Tejas S; Marsh, Graham A; Clark, Alfred; Waugh, Richard E; McGrath, James L

    2014-07-21

    Microfluidic systems are powerful tools for cell biology studies because they enable the precise addition and removal of solutes in small volumes. However, the fluid forces inherent in the use of microfluidics for cell cultures are sometimes undesirable. An important example is chemotaxis systems where fluid flow creates well-defined and steady chemotactic gradients but also pushes cells downstream. Here we demonstrate a chemotaxis system in which two chambers are separated by a molecularly thin (15 nm), transparent, and nanoporous silicon membrane. One chamber is a microfluidic channel that carries a flow-generated gradient while the other chamber is a shear-free environment for cell observation. The molecularly thin membranes provide effectively no resistance to molecular diffusion between the two chambers, making them ideal elements for creating flow-free chambers in microfluidic systems. Analytical and computational flow models that account for membrane and chamber geometry, predict shear reduction of more than five orders of magnitude. This prediction is confirmed by observing the pure diffusion of nanoparticles in the cell-hosting chamber despite high input flow (Q = 10 μL min(-1); vavg ~ 45 mm min(-1)) in the flow chamber only 15 nm away. Using total internal reflection fluorescence (TIRF) microscopy, we show that a flow-generated molecular gradient will pass through the membrane into the quiescent cell chamber. Finally we demonstrate that our device allows us to expose migrating neutrophils to a chemotactic gradient or fluorescent label without any influence from flow.

  1. A silicon-based peptide biosensor for label-free detection of cancer cells

    NASA Astrophysics Data System (ADS)

    Martucci, Nicola M.; Rea, Ilaria; Ruggiero, Immacolata; Terracciano, Monica; De Stefano, Luca; Migliaccio, Nunzia; Dardano, Principia; Arcari, Paolo; Rendina, Ivo; Lamberti, Annalisa

    2015-05-01

    Sensitive and accurate detection of cancer cells plays a crucial role in diagnosis of cancer and minimal residual disease, so being one of the most hopeful approaches to reduce cancer death rates. In this paper, a strategy for highly selective and sensitive detection of lymphoma cells on planar silicon-based biosensor has been evaluated. In this setting an Idiotype peptide, able to specifically bind the B-cell receptor (BCR) of A20 cells in mice engrafted with A20 lymphoma, has been covalently linked to the sensor active surface and used as molecular probe. The biochip here presented showed a coverage efficiency of 85% with a detection efficiency of 8.5×10-3 cells/μm2. The results obtained suggested an efficient way for specific label-free cell detection by using a silicon-based peptide biosensor. In addition, the present recognition strategy, besides being useful for the development of sensing devices capable of monitoring minimal residual disease, could be used to find and characterize new specific receptor-ligand interactions through the screening of a recombinant phage library.

  2. Synthesis of a fluorescently labeled compound for the detection of arsenic-induced apoptotic HL60 cells.

    PubMed

    Femia, A Lis; Temprana, C Facundo; Amor, M Silvia; Grasselli, Mariano; Alonso, Silvia Del V

    2012-03-01

    Arsenic compounds have shown medical usefulness since they proved to be effective in causing complete remission of acute promyelocytic leukemia. In this work we obtained a fluorescently labeled arsenic compound that can be used with current fluorescence techniques for basic and applied research, focused on arsenic-induced apoptosis studies. This compound is an arsanilic acid bearing a covalently linked FITC that was chemically synthesized and characterized by fluorescence, UV-Vis, mass and FTIR spectrometry. In addition, we assessed its apoptotic activity as well as its fluorescent labeling properties in HL60 cell line as a leukemia cell model through flow cytometry. We obtained a compound with a 1:1 FITC:arsenic ratio and a 595 m/z, confirming its structure by FTIR. This compound proved to be useful at inducing apoptosis in the leukemia cell model and labeling this apoptotic cell population, in such a way that the highest FITC fluorescence correlated with the highest arsenic amount.

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

    NASA Astrophysics Data System (ADS)

    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.

  4. Efficient (18)F-Labeling of Synthetic Exendin-4 Analogues for Imaging Beta Cells.

    PubMed

    Keliher, Edmund J; Reiner, Thomas; Thurber, Greg M; Upadhyay, Rabi; Weissleder, Ralph

    2012-08-01

    A number of exendin derivatives have been developed to target glucagon-like peptide 1 (GLP-1) receptors on beta cells in vivo. Modifications of exendin analogues have been shown to have significant effects on pharmacokinetics and, as such, have been used to develop a variety of therapeutic compounds. Here, we show that an exendin-4, modified at position 12 with a cysteine conjugated to a tetrazine, can be labeled with (18)F-trans-cyclooctene and converted into a PET imaging agent at high yields and with good selectivity. The agent accumulates in beta cells in vivo and has sufficiently high accumulation in mouse models of insulinomas to enable in vivo imaging. PMID:23997998

  5. Efficient 18F-Labeling of Synthetic Exendin-4 Analogues for Imaging Beta Cells

    PubMed Central

    Keliher, Edmund J; Reiner, Thomas; Thurber, Greg M; Upadhyay, Rabi; Weissleder, Ralph

    2012-01-01

    A number of exendin derivatives have been developed to target glucagon-like peptide 1 (GLP-1) receptors on beta cells in vivo. Modifications of exendin analogues have been shown to have significant effects on pharmacokinetics and, as such, have been used to develop a variety of therapeutic compounds. Here, we show that an exendin-4, modified at position 12 with a cysteine conjugated to a tetrazine, can be labeled with 18F-trans-cyclooctene and converted into a PET imaging agent at high yields and with good selectivity. The agent accumulates in beta cells in vivo and has sufficiently high accumulation in mouse models of insulinomas to enable in vivo imaging. PMID:23997998

  6. Fast Label-Free Cytoskeletal Network Imaging in Living Mammalian Cells

    PubMed Central

    Bon, Pierre; Lécart, Sandrine; Fort, Emmanuel; Lévêque-Fort, Sandrine

    2014-01-01

    We present a full-field technique that allows label-free cytoskeletal network imaging inside living cells. This noninvasive technique allows monitoring of the cytoskeleton dynamics as well as interactions between the latter and organelles on any timescale. It is based on high-resolution quantitative phase imaging (modified Quadriwave lateral shearing interferometry) and can be directly implemented using any optical microscope without modification. We demonstrate the capability of our setup on fixed and living Chinese hamster ovary cells, showing the cytoskeleton dynamics in lamellipodia during protrusion and mitochondria displacement along the cytoskeletal network. In addition, using the quantitative function of the technique, along with simulation tools, we determined the refractive index of a single tubulin microtubule to be ntubu=2.36±0.6 at λ=527 nm. PMID:24739158

  7. Marked by association: techniques for proximity-dependent labeling of proteins in eukaryotic cells.

    PubMed

    Roux, Kyle J

    2013-10-01

    Various methods have been established for the purpose of identifying and characterizing protein-protein interactions (PPIs). This diverse toolbox provides researchers with options to overcome challenges specific to the nature of the proteins under investigation. Among these techniques is a category based on proximity-dependent labeling of proteins in living cells. These can be further partitioned into either hypothesis-based or unbiased screening methods, each with its own advantages and limitations. Approaches in which proteins of interest are fused to either modifying enzymes or receptor sequences allow for hypothesis-based testing of protein proximity. Protein crosslinking and BioID (proximity-dependent biotin identification) permit unbiased screening of protein proximity for a protein of interest. Here, we evaluate these approaches and their applications in living eukaryotic cells.

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

    PubMed

    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

  9. Tc-99m-labeled red blood cells for the measurement of red cell mass in newborn infants: concise communication

    SciTech Connect

    Linderkamp, O.; Betke, K.; Fendel, H.; Klemm, J.; Lorenzen, K.; Riegel, K.P.

    1980-07-01

    In vitro and in vivo investigations were performed to examine the binding of Tc-99m to neonatal red blood cells (RBC). Labeling efficiency was about 90%, and unbound Tc-99m less than 3% after one washing, in premature and full-term newborns and in children. Thus presence of high percentages of fetal hemoglobin (Hb F) did not influence the labeling of RBCs with Tc-99m. RBCs of 11 newborns were hemolysed and the distribution of Tc-99m on RBC components was analyzed. Although Hb F percentage averaged (60.0 +- 8.10)% (s.d.), only (11.9 +- 3.7)% of Tc-99m was bound by Hb F, whereas (45.0 +- 6.1)% was associated with Hb A. RBC membranes bound (13.7 +- 4.3)% and (29.3 +- 4.0)% were found unbound in hemolysates. These results indicate that Tc-99m preferentially binds to beta chains. In vivo equilibration of Tc-99m RBCs and of albumin labeled with Evans blue was investigated in five newborn infants. Tc-99m RBCs were stable in each case during the first hour after injection. Elution of Tc-99m from RBCs was (3.4 +- 1.5)% per h. Body-to-venous hematocrit ratio averaged 0.86 +- 0.03.

  10. Method and kit for the selective labeling of red blood cells in whole blood with Tc-99m

    DOEpatents

    Srivastava, S.C.; Babich, J.W.; Straub, R.; Richards, P.

    1988-07-05

    Disclosed herein are a method and kit for the preparation of [sup 99m]Tc labeled red blood cells using whole blood in a closed sterile system containing stannous tin in a form such that it will enter the red blood cells and be available for the reduction of technetium. No Drawings

  11. Method and kit for the selective labeling of red blood cells in whole blood with TC-99M

    DOEpatents

    Srivastava, Suresh C.; Babich, John W.; Straub, Rita; Richards, Powell

    1988-01-01

    Disclosed herein are a method and kit for the preparation of .sup.99m Tc labeled red blood cells using whole blood in a closed sterile system containing stannous tin in a form such that it will enter the red blood cells and be available therein for the reduction of technetium.

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

    PubMed

    Sheridan, Erin J; Austin, Christopher J D; Aitken, Jade B; Vogt, Stefan; Jolliffe, Katrina A; Harris, Hugh H; Rendina, Louis M

    2013-03-01

    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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  14. Inhibition of certain strains of HIV-1 by cell surface polyanions in the form of cholesterol-labeled oligonucleotides

    SciTech Connect

    Ahn, Kwang-Soo; Ou, Wu; Silver, Jonathan . E-mail: jsilver@nih.gov

    2004-12-05

    Cholesterol-labeled oligonucleotides were found several years ago to inhibit HIV-1 in tissue culture at nanomolar concentrations. We present evidence that this is mainly due to an electrostatic interaction between polyanionic oligonucleotide concentrated at the cell surface and a positively charged region in the V3 loop of the HIV-1 envelope protein. When added to tissue culture, cholesterol-labeled oligonucleotides became concentrated at the plasma membrane and potently inhibited virus entry and cell fusion mediated by the envelope protein of some X4 strains of HIV-1, but had little effect on fusion mediated by R5 strains of HIV-1, amphotropic MLV envelope protein, or VSV-G protein. Noncholesterol-labeled oligonucleotides did not bind to the cell surface or inhibit fusion. The pattern of susceptibility to cholesterol-labeled oligonucleotides among HIV-1 strains was the same as reported for nonmembrane-associating polyanions such as dextran sulfate, but the cholesterol-labeled oligonucleotides were effective at lower concentrations. Substitution of a basic 33 amino acid V3 loop sequence from the envelope protein of a resistant strain into a susceptible strain made the envelope protein resistant to inhibition. Inhibition by cholesterol-labeled oligonucleotides was abrogated by the polycation DEAE-dextran. Cholesterol-labeled oligonucleotides bound to nonraft regions of the plasma membrane and did not inhibit HIV virus binding to cells. Many infectious agents first associate with target cells via relatively nonspecific charge interactions; our data suggest that molecules that combine a membrane-targeting motif with multiple negative charges might be useful to modify these interactions.

  15. A DNA hybridization system for labeling of neural stem cells with SPIO nanoparticles for MRI monitoring post-transplantation.

    PubMed

    Egawa, Edgar Y; Kitamura, Narufumi; Nakai, Ryusuke; Arima, Yusuke; Iwata, Hiroo

    2015-06-01

    Neural stem cells (NSCs) demonstrate encouraging results in cell replacement therapy for neurodegenerative disorders and traumatic injury in the central nervous system. Monitor the survival and migration of transplanted cells would provide us important information concerning the performance and integration of the graft during the therapy time course. Magnetic resonance imaging (MRI) allow us to monitor the transplanted cells in a non-invasive way. The only requirement is to use an appropriate contrast agent to label the transplanted cells. Superparamagnetic iron oxide (SPIO) nanoparticles are one of the most commonly used contrast agent for MRI detection of transplanted cells. SPIO nanoparticles demonstrated to be suitable for labeling several types of cells including NSCs. However, the current methods for SPIO labeling are non-specific, depending mostly on electrostatic interactions, demanding relatively high SPIO concentration, and long incubation time, which can affect the viability of cells. In this study, we propose a specific and relatively fast method to label NSCs with SPIO nanoparticles via DNA hybridization. Two short single stranded DNAs (ssDNAs), oligo[dT]20 and oligo[dA]20 were conjugated with a lipid molecule and SPIO nanoparticle respectively. The labeling process comprises two simple steps; first the cells are modified to present oligo[dT]20 ssDNA on the cell surface, then the oligo[dA]20 ssDNA conjugated with SPIO nanoparticles are presented to the modified cells to allow the oligo[dT]20-oligo[dA]20 hybridization. The method showed to be non-toxic at concentrations up to 50 μg/mL oligo[dA]20-SPIO nanoparticles. Presence of SPIO nanoparticles at cell surface and cell cytoplasm was verified by transmission electron microscopy (TEM). SPIO labeling via DNA hybridization demonstrated to not interfere on NSCs proliferation, aggregates formation, and differentiation. NSCs labeled with SPIO nanoparticles via DNA hybridization system were successfully

  16. Detection of viability of transplanted beta cells labeled with a novel contrast agent - polyvinylpyrrolidone-coated superparamagnetic iron oxide nanoparticles by magnetic resonance imaging.

    PubMed

    Zhang, Bo; Jiang, Biao; Chen, Ying; Huang, Hai; Xie, Qiuping; Kang, Muxing; Zhang, Hui; Zhai, Chuanxin; Wu, Yulian

    2012-01-01

    Islets can be visualized on MRI by labeling with superparamagnetic contrast agent during the transplantation procedure. However, whether the signal intensity reflects the cell number and cellular viability has not been determined. We used a self-synthesized novel superparamagnetic contrast agent -polyvinylpyrrolidone-coated superparamagnetic iron oxide nanoparticles (PVP-SPIO) - to label β-TC-6 cells (a mouse insulinoma cell line) or primary islets with commercial Feridex as a control. The labeling efficiency of two agents was compared by Prussian blue staining, intracellular iron content determination and MR scanning. Cells were exposed to hypoxia, high-glucose or exogenous H₂O₂ stimulation before/after PVP-SPIO labeling. Normal and injured cells were also transplanted into renal subcapsule. A clinically used 3.0 T MR scan was performed in vitro and 24 h post-transplantation to investigate the correlation between cellular viability and signal. Our PVP-SPIO displayed superior biocompatibility and magnetic properties. All of the cells could be labeled at 100 µg/ml iron concentration after 24 h incubation. At 100 µg/ml iron concentration, 1 × 10⁵ β cells labeled with PVP-SPIO could already be visualized in vitro by MRI, less than the detection threshold of Feridex. There existed a linear correlation between the number of labeled cells and R₂ value on the T₂ -weighted images. The signal intensity and the intracellular iron content declined along with the decreased viability of labeled cells. There was also a significant difference in signal intensity between injured and normal labeled cells after transplantation. From these results, we concluded that PVP-SPIO possessed superior cell labeling efficiency, and β cells could be labeled without compromising viability and function. The signal intensity on MRI might be a useful predictor to evaluate the number and the viability of PVP-SPIO-labeled cells.

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

    PubMed

    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. PMID:27232491

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  20. Superparamagnetic iron oxide nanoparticle-labeled cells as an effective vehicle for tracking the GFP gene marker using magnetic resonance imaging

    PubMed Central

    Zhang, Z; Mascheri, N; Dharmakumar, R; Fan, Z; Paunesku, T; Woloschak, G; Li, D

    2010-01-01

    Background Detection of a gene using magnetic resonance imaging (MRI) is hindered by the magnetic resonance (MR) targeting gene technique. Therefore it may be advantageous to image gene-expressing cells labeled with superparamagnetic iron oxide (SPIO) nanoparticles by MRI. Methods The GFP-R3230Ac (GFP) cell line was incubated for 24 h using SPIO nanoparticles at a concentration of 20 μg Fe/mL. Cell samples were prepared for iron content analysis and cell function evaluation. The labeled cells were imaged using fluorescent microscopy and MRI. Results SPIO was used to label GFP cells effectively, with no effects on cell function and GFP expression. Iron-loaded GFP cells were successfully imaged with both fluorescent microscopy and T2*-weighted MRI. Prussian blue staining showed intracellular iron accumulation in the cells. All cells were labeled (100% labeling efficiency). The average iron content per cell was 4.75±0.11 pg Fe/cell (P<0.05 versus control). Discussion This study demonstrates that the GFP expression of cells is not altered by the SPIO labeling process. SPIO-labeled GFP cells can be visualized by MRI; therefore, GFP, a gene marker, was tracked indirectly with the SPIO-loaded cells using MRI. The technique holds promise for monitoring the temporal and spatial migration of cells with a gene marker and enhancing the understanding of cell- and gene-based therapeutic strategies. PMID:18956269

  1. Optofluidic device for label-free cell classification from whole blood.

    PubMed

    Wu, Tsung-Feng; Mei, Zhe; Lo, Yu-Hwa

    2012-10-01

    We demonstrated a unique optofluidic lab-on-a-chip device that can measure optically encoded forward scattering signals. From the design of the spatial pattern, we can measure the position and velocity of each cell in the flow and generate a 2-D cell distribution plot over the cross section of the channel. Moreover, we have demonstrated that the cell distribution is highly sensitive to its size and stiffness. The latter is an important biomarker for cell classification and our method offers a simple and unequivocal method to classify cells by their size and stiffness. We have proved the concept using live and fixed HeLa cells. Due to the stiffness and size difference of neutrophils compared to other types of white blood cells, we have demonstrated detection of neutrophils from other blood cells. Finally, we have performed the test using 5 μL of human blood. In a greatly simplified blood preparation process, skipping the usual steps of anticoagulation, centrifuge, antibody labelling or staining, filtering, etc., we have demonstrated that our device and detection principle can count neutrophils in whole human blood. Our system is compact, inexpensive and simple to fabricate and operate, having a commodity laser diode and a Si PIN photoreceiver as the main pieces of hardware. Although the results are still preliminary, the studies indicate that this optofluidic device holds promise to be a point-of-care and home care device to measure neutrophil concentration, which is the key indicator of the immune functions for cancer patients undergoing chemotherapy. PMID:22875178

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

    SciTech Connect

    McGuire, Ryan M.; Silberg, Jonathan J.; Pereira, Fred A.; Raphael, Robert M.

    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.

  3. Sialic Acid-Imprinted Fluorescent Core-Shell Particles for Selective Labeling of Cell Surface Glycans.

    PubMed

    Shinde, Sudhirkumar; El-Schich, Zahra; Malakpour, Atena; Wan, Wei; Dizeyi, Nishtman; Mohammadi, Reza; Rurack, Knut; Gjörloff Wingren, Anette; Sellergren, Börje

    2015-11-01

    The expression of cell surface glycans terminating with sialic acid (SA) residues has been found to correlate with various disease states there among cancer. We here report a novel strategy for specific fluorescence labeling of such motifs. This is based on sialic acid-imprinted core-shell nanoparticles equipped with nitrobenzoxadiazole (NBD) fluorescent reporter groups allowing environmentally sensitive fluorescence detection at convenient excitation and emission wavelengths. Imprinting was achieved exploiting a hybrid approach combining reversible boronate ester formation between p-vinylphenylboronic acid and SA, the introduction of cationic amine functionalities, and the use of an NBD-appended urea-monomer as a binary hydrogen-bond donor targeting the SA carboxylic acid and OH functionalities. The monomers were grafted from 200 nm RAFT-modified silica core particles using ethylene glycol dimethacrylate (EGDMA) as cross-linker resulting in a shell thickness of ca. 10 nm. The particles displayed strong affinity for SA in methanol/water mixtures (K = 6.6 × 10(5) M(-1) in 2% water, 5.9 × 10(3) M(-1) in 98% water, B(max) ≈ 10 μmol g(-1)), whereas binding of the competitor glucuronic acid (GA) and other monosaccharides was considerably weaker (K (GA) = 1.8 × 10(3) M(-1) in 98% water). In cell imaging experiments, the particles selectively stained different cell lines in correlation with the SA expression level. This was further verified by enzymatic cleavage of SA and by staining using a FITC labeled SA selective lectin. PMID:26414878

  4. Distribution of injected technetium(99m)-labeled mesenchymal stem cells in horses with naturally occurring tendinopathy.

    PubMed

    Becerra, Patricia; Valdés Vázquez, Miguel A; Dudhia, Jayesh; Fiske-Jackson, Andrew R; Neves, Francisco; Hartman, Neil G; Smith, Roger K W

    2013-07-01

    This study aimed to investigate immediate cell survival and distribution following different administration routes of mesenchymal stem cells (MSCs) into naturally occurring tendon injuries. Ten million MSCs, labeled with technetium-99m hexamethylpropyleneamine oxime, were implanted into 13 horses with naturally occurring tendon or ligament injuries intra-lesionally, intravenously and by regional perfusion, and traced for up to 48 h using planar gamma scintigraphy. Labeling efficiencies varied between 1.8% and 18.5% (mean 9.3%). Cells were retained in the damaged area after intra-lesional administration but only 24% of cells were still present within the tendon after 24 h. After intravenous injection, cells largely distributed to the lung fields, with no detectable cells in the tendon lesions. Significant labeling of the tendon lesions was observed in 11/12 horses following regional perfusion but at a lower level to intra-lesional injection. The highest cell numbers were retained after intra-lesional injection, although with considerable cell loss, while regional perfusion may be a viable alternative for MSC delivery. Cells did not "home" to damaged tendon in large numbers after intravenous administration. Cells were detected in the lungs most frequently after intravascular administration, although with no adverse effects. Low cell retention has important implications for designing effective clinical therapies for human clinical use.

  5. Hepatic cavernous hemangioma: diagnosis with /sup 99m/Tc-labeled red cells and single-photon emission CT

    SciTech Connect

    Brodsky, R.I.; Friedman, A.C.; Maurer, A.H.; Radecki, P.D.; Caroline, D.F.

    1987-01-01

    During the performance of high-resolution real-time abdominal sonography, small echogenic hepatic masses are frequently discovered. A second imaging test to confirm the suspected diagnosis of hemangioma is often required. Planar labeled red-cell imaging will often not detect hemangiomas smaller than 3 cm. We studied 14 patients with labeled red-cell scintigraphy and single-photon emission CT (SPECT). Six hemangiomas were diagnosed by SPECT that would have been missed by planar imaging alone. All six were smaller than 2.5 cm. With the addition of SPECT, labeled red-cell scintigraphy has specificity and sensitivity that make it at least as reliable as dynamic CT for the noninvasive diagnosis of hepatic cavernous hemangioma.

  6. Label-retaining assay enriches tumor-initiating cells in glioblastoma spheres cultivated in serum-free medium

    PubMed Central

    Zeng, Lingcheng; Zhao, Yiqing; Ouyang, Taohui; Zhao, Tianyuan; Zhang, Suojun; Chen, Jian; Yu, Jiasheng; Lei, Ting

    2016-01-01

    Label-retaining cells, which are characterized by dormancy or slow cycling, may be identified in a number of human normal and cancer tissues, and these cells demonstrate stem cell potential. In glioblastoma, label-retaining assays to enrich glioma stem cells remain to be fully investigated. In the present study, glioblastoma sphere cells cultured in serum-free medium were initially stained with the cell membrane fluorescent marker DiI. The fluorescence intensity during cell proliferation and sphere reformation was observed. At 2 weeks, the DiI-retaining cells were screened by fluorescence-activated cell sorting and compared phenotypically with the DiI-negative cells in terms of in vitro proliferation, clonogenicity and multipotency and for in vivo tumorigenicity, as well as sensitivity to irradiation and temozolomide treatment. It was observed that DiI-retaining cells accounted for a small proportion, <10%, within the glioblastoma spheres and that DiI-retaining cells proliferated significantly more slowly compared with DiI-negative cells (P=0.011, P=0.035 and P=0.023 in the of NCH421k, NCH441 and NCH644 glioblastoma sphere cell lines). Significantly increased clonogenicity (P=0.002, P=0.034 and P=0.016 in the NCH441, NCH644 and NCH421k glioblastoma sphere cell lines) and three-lineage multipotency were observed in DiI-retaining cells in vitro compared with DiI-negative cells. As few as 100 DiI-retaining cells were able to effectively generate tumors in the immunocompromised mouse brain, whereas the same number of DiI-negative cells possessed no such ability, indicating the increased tumorigenicity of DiI-retaining cells compared with DiI-negative cells. Furthermore, DiI-retaining cells demonstrated significant resistance following irradiation (P=0.012, P=0.024 and P=0.036) and temozolomide (P=0.003, P=0.005 and P=0.029) compared with DiI-negative cells in the NCH421k, NCH441 and NCH644 glioblastoma sphere cell lines, respectively. It was concluded that label

  7. Color transformation and fluorescence of Prussian blue-positive cells: implications for histologic verification of cells labeled with superparamagnetic iron oxide nanoparticles.

    PubMed

    Frank, Joseph A; Kalish, Heather; Jordan, E Kay; Anderson, Stasia A; Pawelczyk, Edyta; Arbab, Ali S

    2007-01-01

    Superparamagnetic iron oxide (SPIO) nanoparticles, either modified or in combination with other macromolecules, are being used for magnetic labeling of stem cells and other cells to monitor cell trafficking by magnetic resonance imaging (MRI) in experimental models. The correlation of histology to MRI depends on the ability to detect SPIO-labeled cells using Prussian blue (PB) stain and fluorescent tags to cell surface markers. Exposure of PB-positive sections to ultraviolet light at a wavelength of 365 nm commonly used fluorescence microscopy can result in color transformation of PB-positive material from blue to brown. Although the PB color transformation is primarily an artifact that may occur during fluorescence microscopy, the transformation can be manipulated using imaging process software for the detection of low levels of iron labeled cells in tissues samples.

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

    PubMed

    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

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

    PubMed

    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.

  10. Silica Encapsulated Gold Nanoparticles as SERS Labels for the Detection of Lymphoma B-Cells in Tissue Sections

    NASA Astrophysics Data System (ADS)

    Al-Faouri, Tamara

    The surface of silica encapsulated gold nanoparticles with trans-1,2-bis (4-pyridyl) ethylene Raman active dye were utilized as SERS labels to target CD20 surface protein on lymphoma B-cells in human tissue sections with CLL or FL. SERS labels were functionalized with various antibody linkers including carboxylic, aldehyde, and heterobifunctional PEG chains with an NHS end, to permit them to bind to tissue section samples. NP samples and tissue sections were characterized through UV-Vis spectroscopy, TEM, XPS, Zeta potential measurements, Dark Field microscopy, Raman spectroscopy, NMR, and AFM. The number of SERS labels present on a tissue sample was estimated using dark field images and a particle counting software. It was found that the heterobifunctional PEG chains linker provided the most specific binding of SERS labels with an estimated NP count of 1.33x106 NPs on the whole tissue and produced the highest Raman scatter intensity of approximately 48600 counts.

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

    PubMed Central

    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. PMID:24451999

  12. Sterically shielded spin labels for in-cell EPR spectroscopy: analysis of stability in reducing environment.

    PubMed

    Jagtap, A P; Krstic, I; Kunjir, N C; Hänsel, R; Prisner, T F; Sigurdsson, S Th

    2015-01-01

    Electron paramagnetic resonance (EPR) spectroscopy is a powerful and widely used technique for studying structure and dynamics of biomolecules under bio-orthogonal conditions. In-cell EPR is an emerging area in this field; however, it is hampered by the reducing environment present in cells, which reduces most nitroxide spin labels to their corresponding diamagnetic N-hydroxyl derivatives. To determine which radicals are best suited for in-cell EPR studies, we systematically studied the effects of substitution on radical stability using five different classes of radicals, specifically piperidine-, imidazolidine-, pyrrolidine-, and isoindoline-based nitroxides as well as the Finland trityl radical. Thermodynamic parameters of nitroxide reduction were determined by cyclic voltammetry; the rate of reduction in the presence of ascorbate, cellular extracts, and after injection into oocytes was measured by continuous-wave EPR spectroscopy. Our study revealed that tetraethyl-substituted nitroxides are good candidates for in-cell EPR studies, in particular pyrrolidine derivatives, which are slightly more stable than the trityl radical. PMID:25348344

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

    NASA Astrophysics Data System (ADS)

    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.

  14. Functional investigations on human mesenchymal stem cells exposed to magnetic fields and labeled with clinically approved iron nanoparticles

    PubMed Central

    2010-01-01

    Background For clinical applications of mesenchymal stem cells (MSCs), labeling and tracking is crucial to evaluate cell distribution and homing. Magnetic resonance imaging (MRI) has been successfully established detecting MSCs labeled with superparamagnetic particles of iron oxide (SPIO). Despite initial reports that labeling of MSCs with SPIO is safe without affecting the MSC's biology, recent studies report on influences of SPIO-labeling on metabolism and function of MSCs. Exposition of cells and tissues to high magnetic fields is the functional principle of MRI. In this study we established innovative labeling protocols for human MSCs using clinically established SPIO in combination with magnetic fields and investigated on functional effects (migration assays, quantification of colony forming units, analyses of gene and protein expression and analyses on the proliferation capacity, the viability and the differentiation potential) of magnetic fields on unlabeled and labeled human MSCs. To evaluate the imaging properties, quantification of the total iron load per cell (TIL), electron microscopy, and MRI at 3.0 T were performed. Results Human MSCs labeled with SPIO permanently exposed to magnetic fields arranged and grew according to the magnetic flux lines. Exposure of MSCs to magnetic fields after labeling with SPIO significantly enhanced the TIL compared to SPIO labeled MSCs without exposure to magnetic fields resulting in optimized imaging properties (detection limit: 1,000 MSCs). Concerning the TIL and the imaging properties, immediate exposition to magnetic fields after labeling was superior to exposition after 24 h. On functional level, exposition to magnetic fields inhibited the ability of colony formation of labeled MSCs and led to an enhanced expression of lipoprotein lipase and peroxisome proliferator-activated receptor-γ in labeled MSCs under adipogenic differentiation, and to a reduced expression of alkaline phosphatase in unlabeled MSCs under

  15. Phosphorylcholine-Coated Semiconducting Polymer Nanoparticles as Rapid and Efficient Labeling Agents for in vivo Cell Tracking

    PubMed Central

    Pu, Kanyi; Shuhendler, Adam J.; Valta, Maija P.; Cui, Lina; Saar, Matthias; Peehl, Donna M.

    2014-01-01

    Despite the pressing need to noninvasively monitor transplanted cells in vivo with fluorescence imaging, desirable fluorescent agents with rapid labeling capability, durable brightness, and ideal biocompatibility remain lacking. Herein we report phosphorylcholine-coated near-infrared (NIR) fluorescent semiconducting polymer nanoparticles (SPNs) as a new class of rapid, efficient and cytocompatible labeling nanoagents for in vivo cell tracking. The phosphorylcholine coating results in efficient and rapid endocytosis and allows the SPN to enter cells within 0.5 h in complete culture medium apparently independent of the cell type, while its NIR fluorescence leads to a tissue penetration depth of 0.5 cm. In comparison to quantum dots and Cy5.5, the SPN is tolerant to physiologically ubiquitous reactive oxygen species ROS, resulting in durable fluorescence both in vitro and in vivo. These desirable physical and physiological properties of the SPN permit cell tracking of human renal cell carcinoma (RCC) cells in living mice at a lower limit of detection of 10,000 cells with no obvious alteration of cell phenotype after 12 days. SPNs thus could provide unique opportunities for optimizing cellular therapy and deciphering pathological processes as a cell tracking label. PMID:24668903

  16. Imaging of nanoparticle-labeled stem cells using magnetomotive optical coherence tomography, laser speckle reflectometry, and light microscopy

    NASA Astrophysics Data System (ADS)

    Cimalla, Peter; Werner, Theresa; Winkler, Kai; Mueller, Claudia; Wicht, Sebastian; Gaertner, Maria; Mehner, Mirko; Walther, Julia; Rellinghaus, Bernd; Wittig, Dierk; Karl, Mike O.; Ader, Marius; Funk, Richard H. W.; Koch, Edmund

    2015-03-01

    Cell transplantation and stem cell therapy are promising approaches for regenerative medicine and are of interest to researchers and clinicians worldwide. However, currently, no imaging technique that allows three-dimensional in vivo inspection of therapeutically administered cells in host tissues is available. Therefore, we investigate magnetomotive optical coherence tomography (MM-OCT) of cells labeled with magnetic particles as a potential noninvasive cell tracking method. We develop magnetomotive imaging of mesenchymal stem cells for future cell therapy monitoring. Cells were labeled with fluorescent iron oxide nanoparticles, embedded in tissue-mimicking agar scaffolds, and imaged using a microscope setup with an integrated MM-OCT probe. Magnetic particle-induced motion in response to a pulsed magnetic field of 0.2 T was successfully detected by OCT speckle variance analysis, and cross-sectional and volumetric OCT scans with highlighted labeled cells were obtained. In parallel, fluorescence microscopy and laser speckle reflectometry were applied as two-dimensional reference modalities to image particle distribution and magnetically induced motion inside the sample, respectively. All three optical imaging modalities were in good agreement with each other. Thus, magnetomotive imaging using iron oxide nanoparticles as cellular contrast agents is a potential technique for enhanced visualization of selected cells in OCT.

  17. The labeling of stem cells by superparamagnetic iron oxide nanoparticles modified with PEG/PVP or PEG/PEI.

    PubMed

    Yang, Gao; Ma, Weiqiong; Zhang, Baolin; Xie, Qi

    2016-05-01

    Poly(ethylene glycol) (PEG) and poly(vinyl pyrrolidone) (PVP) co-modified superparamagnetic iron oxide nanoparticles (SPIONs) (PEG/PVP-SPIONs), and PEG and poly(ethylene imine) (PEI) co-modified SPIONs (PEG/PEI-SPIONs) synthesized by thermal decomposition have been used as magnetic resonance imaging (MRI) contrast agents to label adipose-derived stem cells (ADSCs). Efficient cell labeling was achieved after incubation with PEG/PVP-SPIONs and PEG/PEI-SPIONs for 12h, and the MRI of labeled cells was evaluated. The cell viability tests showed the low cytotoxicity of PEG/PVP-SPIONs and PEG/PEI-SPIONs. The cellular iron content incubated with PEG/PVP-SPIONs at a concentration of 25 μg/ml was 6.96 pg/cell, the cellular iron contents incubated with PEG/PEI-SPIONs at concentrations of 12 and 25 μg/ml were 20.16, 35.4 pg/cell, respectively. The SPIONs were located predominantly in the intracellular vesicles. The cellular iron oxide uptake was significantly high after incubation with PEG/PEI-SPIONs as compared with the commercial iron oxide agents (Feridex, Feridex@PLL, Resovist and Resovist@PLL) reported. This work demonstrates that PEG/PEI-SPIONs are the competent agents for the labeling of ADSCs. PMID:26952437

  18. Selective Label-free Electrokinetic Cell Tracker (SELECT): a novel liquid platform for cell characterization

    NASA Astrophysics Data System (ADS)

    Taruvai Kalyana Kumar, Rajeshwari; de Mello Gindri, Izabelle; Kinnamon, David; Kanchustambham, Pradyotha; Rodrigues, Danieli; Prasad, Shalini; BiomaterialsOsseointegration; Novel Engineering Lab Collaboration

    2015-03-01

    Characterization and analysis of rare cells provide critical cues for early diagnosis of diseases. Electrokinetic cell separation has been previously established to have greater efficiency when compared to traditional flow cytometry methods. It has been shown by many researchers that buffer solutions in which cells are suspended in, have enormous effects on producing required dielectrophoretic (DEP) forces to characterize cells. Most commonly used suspension buffers used are deionized water and cell media. However, these solutions exhibit high level of intrinsic noise, which greatly masks the electrokinetic signals from cells under study. Ionic liquids (ILs) show promise towards the creation of conductive fluids with required electrical properties. The goal of this project is to design and test ILs for enhancing DEP forces on cells while creating an environment for preserving their integrity. We analyzed two methylimidazolium based ILs as suspension medium for cell separation. These dicationic ILs possess slight electrical and structural differences with high thermal stability. The two ILs were tested for cytotoxicity using HeLa and bone cells. The effects of electrical neutrality, free charge screening due to ILs towards enhanced electrokinetic signals from cells were studied with improved system resolution and no harmful effects.

  19. Label-Free Cell Phenotypic Identification of D-Luciferin as an Agonist for GPR35.

    PubMed

    Hu, Heidi; Deng, Huayun; Fang, Ye

    2016-01-01

    D-Luciferin (also known as beetle or firefly luciferin) is one of the most widely used bioluminescent reporters for monitoring in vitro or in vivo luciferase activity. The identification of several natural phenols and thieno[3,2-b]thiophene-2-carboxylic acid derivatives as agonists for GPR35, an orphan G protein-coupled receptor, had motivated us to examine the pharmacological activity of D-Luciferin, given that it also contains phenol and carboxylic acid moieties. Here, we describe label-free cell phenotypic assays that ascertain D-Luciferin as a partial agonist for GPR35. The agonistic activity of D-Luciferin at the GPR35 shall evoke careful interpretation of biological data when D-Luciferin or its analogues are used as probes. PMID:27424891

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27339882

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

    PubMed

    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.

  3. Identification of epithelial label-retaining cells at the transition between the anal canal and the rectum in mice

    PubMed Central

    Runck, Laura A; Kramer, Megan; Ciraolo, Georgianne; Lewis, Alfor G

    2010-01-01

    In certain regions of the body, transition zones exist where stratified squamous epithelia directly abut against other types of epithelia. Certain transition zones are especially prone to tumorigenesis an example being the anorectal junction, although the reason for this is not known. One possibility is that the abrupt transition of the simple columnar epithelium of the colon to the stratified squamous epithelium of the proximal portion of the anal canal may contain a unique stem cell niche. We investigated whether the anorectal region contained cells with stem cell properties relative to the adjacent epithelium. We utilized a tetracycline-regulatable histone H2B-GFP transgenic mice model, previously used to identify hair follicle stem cells, to fluorescently label slow-cycling anal epithelial cells (e.g., prospective stem cells) in combination with a panel of putative stem cell markers. We identified a population of long-term GFP label-retaining cells concentrated at the junction between the anal canal and the rectum. These cells are BrdU-retaining cells and expressed the stem cell marker CD34. Moreover, tracking the fate of the anal label-retaining cells in vivo revealed that the slow-cycling cells only gave rise to progeny of the anal epithelium. In conclusion, we identified a unique population of cells at the anorectal junction which can be separated from the other basal anal epithelial cells based upon the expression of the stem cell marker CD34 and integrin α6, and thus represent a putative anal stem cell population. PMID:20647777

  4. In vivo Tracking of Mesenchymal Stem Cells Labeled with a Novel Chitosan-coated Superparamagnetic Iron Oxide Nanoparticles using 3.0T MRI

    PubMed Central

    Reddy, Alavala Matta; Shim, Hyung Jin; Ahn, Chiyoung; Lee, Hyo Sook; Suh, Yong Jae; Park, Eon Sub

    2010-01-01

    This study aimed to characterize and MRI track the mesenchymal stem cells labeled with chitosan-coated superparamagnetic iron oxide (Chitosan-SPIO). Chitosan-SPIO was synthesized from a mixture of FeCl2 and FeCl3. The human bone marrow derived mesenchymal stem cells (hBM-MSC) were labeled with 50 µg Fe/mL chitosan-SPIO and Resovist. The labeling efficiency was assessed by iron content, Prussian blue staining, electron microscopy and in vitro MR imaging. The labeled cells were also analyzed for cytotoxicity, phenotype and differentiation potential. Electron microscopic observations and Prussian blue staining revealed 100% of cells were labeled with iron particles. MR imaging was able to detect the labeled MSC successfully. Chitosan-SPIO did not show any cytotoxicity up to 200 µg Fe/mL concentration. The labeled stem cells did not exhibit any significant alterations in the surface markers expression or adipo/osteo/chondrogenic differentiation potential when compared to unlabeled control cells. After contralateral injection into rabbit ischemic brain, the iron labeled stem cells were tracked by periodical in vivo MR images. The migration of cells was also confirmed by histological studies. The novel chitosan-SPIO enables to label and track MSC for in vivo MRI without cellular alteration. PMID:20119572

  5. A NIR-remote controlled upconverting nanoparticle: an improved tool for living cell dye-labeling

    NASA Astrophysics Data System (ADS)

    Zheng, Bin; Gong, Xiaoqun; Wang, Hanjie; Wang, Sheng; Wang, Huiquan; Li, Wei; Tan, Jian; Chang, Jin

    2015-10-01

    In living cells, due to the selective permeability and complicated cellular environment, the uptake efficiency and fluorescence decay of organic dyes during dye-labeling may be influenced, which may eventually result in poor fluorescent imaging. In this work, a protocol of UCNs@mSiO2-(FA and Azo) core-shell nanocarriers was designed and prepared successfully. The core-shell nanocarriers were assembled from two parts, including a mesoporous silica shell surface modified by folate (FA) and azobenzene (Azo), and an upconverting nanocrystal (UCN) core. The mesoporous silica shell is used for loading organic dyes and conjugating folate which helps to enhance the cellular uptake of nanocarriers. The UCN core works as a transducer to convert near infrared (NIR) light to local UV and visible light to activate a back-and-forth wagging motion of azobenzene molecules on the surface, while the azobenzene acts as a molecular impeller for propelling the release of organic dyes. The nanocarriers of loading organic dyes can maintain the stability of the fluorescent imaging effect better than free organic dyes. The experimental results show that with the help of the nanoparticle, cell uptake efficiency of the model dyes of rhodamine and 4‧, 6-diamidino-2-phenylindole (DAPI) was significantly improved. The release of dyes can only be triggered by NIR light exposure and their quantity is highly dependent on the duration of NIR light exposure, thus realizing NIR-regulated dye release spatiotemporally. Our work may open a novel avenue for precisely controlling UCN-based living cell imaging in biotechnology and diagnostics, as well as studying cell dynamics, cell-cell interactions, and tissue morphogenesis.

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

    NASA Astrophysics Data System (ADS)

    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

  7. 13C NMR studies of gluconeogenesis in rat liver cells: Utilization of labeled glycerol by cells from euthyroid and hyperthyroid rats

    PubMed Central

    Cohen, S. M.; Ogawa, S.; Shulman, R. G.

    1979-01-01

    The gluconeogenic pathway from [2-13C]glycerol and [1,3-13C]glycerol has been followed in suspensions of isolated rat hepatocytes at 25°C by 13C NMR at 90.5 MHz. The flow of label through the major pathway from glycerol to L-glycerol 3-phosphate and into glucose was followed in cells from control and triiodothyronine-treated rats. Treatment increased the rates of glucose formation and glycerol consumption 2-fold and decreased the αGP level to 40%. We calculate that ≈60% of the flux is through the mitochondrial glycerol phosphate dehydrogenase in cells from triiodothyronine-treated rats, compared with ≈15% in cells from the controls. Equal distribution of label between the trioses of glucose was obtained and, because the C3-C4 spin-spin coupling gives the distribution of labeled carbons in the same molecule, it was possible to measure the amount of triose from unlabeled fructose incorporated into the glucose labeled at carbons 1, 3, 4, and 6. About 10% of the hexoses had flowed through the pentose cycle and back into the hexose pathway in cells from fasted rats. From the distribution of label at glucose carbons not labeled via the major pathway and from the carbon spin-spin splitting patterns observed, we conclude that transketolase is reversible whereas transaldolase is essentially irreversible in the nonoxidative pentose branch. PMID:287001

  8. The effect of varying type and volume of sedimenting agents on leukocyte harvesting and labelling in sickle cell patients.

    PubMed

    Webber, D; Nunan, T O; O'Doherty, M J

    1994-09-01

    Leukocyte labelling in patients with sickle cell anaemia has been reported as difficult if not impossible due to the slow erythrocyte sedimentation rate (ESR) in these patients. This study investigated standard sedimentation methods in patients with sickle cell disease (n = 16) and compared the results obtained with those following changes in the amount and type of sedimenting agent used. Labelling with either 111In-oxine or 99Tcm-exametazime was attempted in only five patients. Replacement of the commonly used 6% Hetastarch (Hespan) with Dextran or Haemaccel did not improve leukocyte harvesting, even when the proportions used of these agents were increased. In most cases where standard procedures for leukocyte collection did not lead to harvesting of viable samples, it was possible to obtain adequate leukocyte labelling in the majority of sickle cell patients using a minor modification of standard techniques. In this group of patients a ratio of 8 ml of Hespan to 16 ml of blood should be used for cell separation. If this fails then donor cells, anti-granulocyte antibody labelling or HIG should be considered.

  9. Visualizing Quantum Dot Labeled ORAI1 Proteins in Intact Cells Via Correlative Light and Electron Microscopy.

    PubMed

    Peckys, Diana B; Alansary, Dalia; Niemeyer, Barbara A; de Jonge, Niels

    2016-08-01

    ORAI1 proteins are ion channel subunits and the essential pore-forming units of the calcium release-activated calcium channel complex essential for T-cell activation and many other cellular processes. In this study, we used environmental scanning electron microscopy (ESEM) with scanning transmission electron microscopy (STEM) detection to image plasma membrane expressed ORAI1 proteins in whole Jurkat T cells in the liquid state. Utilizing a stably transfected Jurkat T cell clone expressing human ORAI1 with an extracellular human influenza hemagglutinin (HA) tag we investigated if liquid-phase STEM can be applied to detect recombinant surface expressed protein. Streptavidin coated quantum dots were coupled in a one-to-one stoichiometry to ORAI1 proteins detected by biotinylated anti-HA fragmented antibody fragments. High-resolution electron microscopic images revealed the individual label locations from which protein pair distances were determined. These data were analyzed using the pair correlation function and, in addition, an analysis of cluster size and frequency was performed. ORAI1 was found to be present in hexamers in a small fraction only, and ORAI1 resided mostly in monomers and dimers.

  10. Gold nanoparticle-packed microdisks for multiplex Raman labelling of cells.

    PubMed

    Zhang, Peipei; Xia, Junfei; Wang, Zhibin; Guan, Jingjiao

    2014-08-01

    Micro/nanoparticles containing densely packed gold nanoparticles (AuNPs) possess unique properties potentially useful for various biomedical applications. The micro/nanoparticles are conventionally produced by the bottom-up methods, which have limited capability for controlling the particle size, shape and structure. This article reports development of a top-down method that integrates layer-by-layer assembly and microcontact printing to fabricate disk-shaped microparticles named microdisks composed of densely packed AuNPs. This method allows precise control of not only the size, shape and structure of the microdisks but also the amount of the AuNPs in the microdisks. The microdisks can be loaded with different Raman reporters to generate characteristic surface-enhanced Raman scattering spectra under the near infrared excitation over a centimetre-scale lens-sample distance. Moreover, the microdisks can be attached to single live cells. This microdisk platform holds potential for multiplex Raman labelling of therapeutic cells for in vivo tracking of the cells.

  11. Label-free imaging of Schwann cell myelination by third harmonic generation microscopy

    PubMed Central

    Lim, Hyungsik; Sharoukhov, Denis; Kassim, Imran; Zhang, Yanqing; Salzer, James L.; Melendez-Vasquez, Carmen V.

    2014-01-01

    Understanding the dynamic axon–glial cell interaction underlying myelination is hampered by the lack of suitable imaging techniques. Here we demonstrate third harmonic generation microscopy (THGM) for label-free imaging of myelinating Schwann cells in live culture and ex vivo and in vivo tissue. A 3D structure was acquired for a variety of compact and noncompact myelin domains, including juxtaparanodes, Schmidt–Lanterman incisures, and Cajal bands. Other subcellular features of Schwann cells that escape traditional optical microscopies were also visualized. We tested THGM for morphometry of compact myelin. Unlike current methods based on electron microscopy, g-ratio could be determined along an extended length of myelinated fiber in the physiological condition. The precision of THGM-based g-ratio estimation was corroborated in mouse models of hypomyelination. Finally, we demonstrated the feasibility of THGM to monitor morphological changes of myelin during postnatal development and degeneration. The outstanding capabilities of THGM may be useful for elucidation of the mechanism of myelin formation and pathogenesis. PMID:25453108

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

    NASA Astrophysics Data System (ADS)

    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.

  13. Rapid Covalent Fluorescence Labeling of Membrane Proteins on Live Cells via Coiled-Coil Templated Acyl Transfer.

    PubMed

    Reinhardt, Ulrike; Lotze, Jonathan; Mörl, Karin; Beck-Sickinger, Annette G; Seitz, Oliver

    2015-10-21

    Fluorescently labeled proteins enable the microscopic imaging of protein localization and function in live cells. In labeling reactions targeted against specific tag sequences, the size of the fluorophore-tag is of major concern. The tag should be small to prevent interference with protein function. Furthermore, rapid and covalent labeling methods are desired to enable the analysis of fast biological processes. Herein, we describe the development of a method in which the formation of a parallel coiled coil triggers the transfer of a fluorescence dye from a thioester-linked coil peptide conjugate onto a cysteine-modified coil peptide. This labeling method requires only small tag sequences (max 23 aa) and occurs with high tag specificity. We show that size matching of the coil peptides and a suitable thioester reactivity allow the acyl transfer reaction to proceed within minutes (rather than hours). We demonstrate the versatility of this method by applying it to the labeling of different G-protein coupled membrane receptors including the human neuropeptide Y receptors 1, 2, 4, 5, the neuropeptide FF receptors 1 and 2, and the dopamine receptor 1. The labeled receptors are fully functional and able to bind the respective ligand with high affinity. Activity is not impaired as demonstrated by activation, internalization, and recycling experiments. PMID:26367072

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

    PubMed Central

    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

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

    PubMed

    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

  16. Impact of magnetic labeling on human and mouse stem cells and their long-term magnetic resonance tracking in a rat model of Parkinson disease.

    PubMed

    Stroh, Albrecht; Boltze, Johannes; Sieland, Katharina; Hild, Katharina; Gutzeit, Cindy; Jung, Tobias; Kressel, Jenny; Hau, Susann; Reich, Doreen; Grune, Tilman; Zimmer, Claus

    2009-01-01

    Magnetic resonance imaging (MRI) of magnetically labeled stem cells has become a valuable tool in the understanding and evaluation of experimental stem cell-based therapies of degenerative central nervous system disorders. This comprehensive study assesses the impact of magnetic labeling of both human and rodent stem cell-containing populations on multiple biologic parameters as maintenance of stemness and oxidative stress levels. Cells were efficiently magnetically labeled with very small superparamagnetic iron oxide particles. Only under the condition of tailored labeling strategies can the impact of magnetic labeling on vitality, proliferation, pluripotency, and oxidative stress levels be minimized. In a rat model of Parkinson disease, magnetically labeled mouse embryonic stem cells were tracked by high-field MRI for 6 months. Significant interindividual differences concerning the spatial distribution of cells became evident. Histologically, transplanted green fluorescent protein-positive iron oxide-labeled cells were clearly identified. No significant increase in oxidative stress levels at the implantation site and no secondary uptake of magnetic label by host phagocytotic cells were observed. Our study strongly suggests that molecular MRI approaches must be carefully tailored to the respective cell population to exert minimal physiologic impact, ensuring the feasibility of this imaging approach for clinical applications.

  17. Tracking Single Cells in Live Animals Using a Photoconvertible Near-Infrared Cell Membrane Label

    PubMed Central

    Wu, Juwell; Runnels, Judith M.; Turcotte, Raphaël; Celso, Cristina Lo; Scadden, David T.; Strom, Terry B.; Lin, Charles P.

    2013-01-01

    We describe a novel photoconversion technique to track individual cells in vivo using a commercial lipophilic membrane dye, DiR. We show that DiR exhibits a permanent fluorescence emission shift (photoconversion) after light exposure and does not reacquire the original color over time. Ratiometric imaging can be used to distinguish photoconverted from non-converted cells with high sensitivity. Combining the use of this photoconvertible dye with intravital microscopy, we tracked the division of individual hematopoietic stem/progenitor cells within the calvarium bone marrow of live mice. We also studied the peripheral differentiation of individual T cells by tracking the gain or loss of FoxP3-GFP expression, a marker of the immune suppressive function of CD4+ T cells. With the near-infrared photoconvertible membrane dye, the entire visible spectral range is available for simultaneous use with other fluorescent proteins to monitor gene expression or to trace cell lineage commitment in vivo with high spatial and temporal resolution. PMID:23990881

  18. Live cell and immuno-labeling techniques to study gravitational effects on single plant cells.

    PubMed

    Chebli, Youssef; Geitmann, Anja

    2015-01-01

    The constant force of gravity plays a primordial role in the ontogeny of all living organisms. Plants, for example, develop their roots and shoots in accordance with the direction of the gravitational vector. Any change in the magnitude and/or the direction of gravity has an important impact on the development of tissues and cells. In order to understand how the gravitational force affects plant cell growth and differentiation, we established two complementary experimental procedures with which the effect of hyper-gravity on single plant cell development can be assessed. The single model cell system we used is the pollen tube or male gametophyte which, because of its rapid growth behavior, is known for its instant response to external stresses. The physiological response of the pollen tube can be assessed in a quantitative manner based on changes in the composition and spatial distribution of its cell wall components and in the precisely defined pattern of its very dynamic cytoplasmic streaming. Here, we provide a detailed description of the steps required for the immuno-localization of various cell wall components using microwave-assisted techniques and we explain how live imaging of the intracellular traffic can be achieved under hyper-gravity conditions.

  19. A novel PET imaging using ⁶⁴Cu-labeled monoclonal antibody against mesothelin commonly expressed on cancer cells.

    PubMed

    Kobayashi, Kazuko; Sasaki, Takanori; Takenaka, Fumiaki; Yakushiji, Hiromasa; Fujii, Yoshihiro; Kishi, Yoshiro; Kita, Shoichi; Shen, Lianhua; Kumon, Hiromi; Matsuura, Eiji

    2015-01-01

    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 (64)Cu 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 (64)Cu-labeled 11-25 mAb highly accumulated in MSLN-expressing tumors as compared to MSLN-negative ones. The (64)Cu-labeled 11-25 mAb is potentially useful as a PET probe capable of being used for wide range of tumors, rather than (18)F-FDG that occasionally provides nonspecific accumulation into the inflammatory lesions.

  20. Positively charged nanogold label allows the observation of fine cell filopodia and flagella in solution by atmospheric scanning electron microscopy.

    PubMed

    Nishiyama, Hidetoshi; Teramoto, Kanae; Suga, Mitsuo; Sato, Chikara

    2014-02-01

    Optical microscopy is generally the first choice to observe microbes and cells. However, its resolution is not always sufficient to reveal specific target structures, such as flagella and pili, which are only nanometers wide. ASEM is an attractive higher resolution alternative, as the sample is observed in aqueous solution at atmospheric pressure. Sample pretreatment for ASEM only comprises simple tasks including fixation, gold labeling, and reagent exchange, taking less than 1 h in total. The lengthy sample pretreatments often required for more classical electron microscopies, such as embedding and dehydration, are unnecessary, and native morphology is preserved. In this study, positively charged nanogold particles were used to label the surfaces of bacteria and cultured animal cells, exploiting their net negative surface charge. After gold enhancement to increase the size of the nanogold particles, ASEM imaging of the bacteria in aqueous solution revealed pili and delicate spiral flagella. This natural shape contrasts starkly with images of dried flagella recorded by standard SEM. Positively charged nanogold labeled the plasma membrane of cultured COS7 cells, and after enhancement allowed filopodia as thin as 100 nm in diameter to be clearly visualized. Based on these studies, ASEM combined with positively charged nanogold labeling promises to become an important tool for the study of cell morphology and dynamics in the near future.

  1. Quaternized carbon dot-modified graphene oxide for selective cell labelling--controlled nucleus and cytoplasm imaging.

    PubMed

    Datta, K K R; Kozák, O; Ranc, V; Havrdová, M; Bourlinos, A B; Safářová, K; Holá, K; Tománková, K; Zoppellaro, G; Otyepka, M; Zbořil, R

    2014-09-25

    Cationic quaternized carbon dots (QCDs) and anionic graphene oxide sheets (GO) are combined via non-covalent interactions following a self-assembly pathway to form highly biocompatible and fluorescent hybrid materials. These hybrids act as selective probes with controlled labelling of the cell nucleus or cytoplasm depending on the QCD loading.

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

    PubMed Central

    Kobayashi, Kazuko; Sasaki, Takanori; Takenaka, Fumiaki; Yakushiji, Hiromasa; Fujii, Yoshihiro; Kishi, Yoshiro; Kita, Shoichi; Shen, Lianhua; Kumon, Hiromi; Matsuura, Eiji

    2015-01-01

    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. PMID:25883990

  3. Localization of obscure gastrointestinal bleeding by technetium 99m-labeled red blood cell scintigraphy.

    PubMed

    Wang, C S; Tzen, K Y; Huang, M J; Wang, J Y; Chen, M F

    1992-01-01

    When a bleeding source from the gastrointestinal (GI) tract cannot be identified with conventional diagnostic studies, it is known as GI bleeding of an obscure origin. In the past three years, in vivo Technetium 99m-labeled red blood cell scintigraphy (RBC scan) has been added to our armamentarium for the diagnosis of obscure GI bleeding. Out of a total of 26 cases, the bleeders could be detected in 12 or 46.2% by RBC scan. The time required ranged from 15 minutes to 24 hours (median, one hour). In 14 patients with active bleeding during the scan period, 11 had positive scans (sensitivity, 78.6%). In 12 patients with inactive bleeding, 11 had negative scans (specificity, 91.7%). Angiography was conducted in nine cases, with all showing negative findings; however, six of them had a positive focus by RBC scan. Laparotomy was performed in seven scan-positive patients, and in three scan-negative patients because of a positive Meckel's scan (two cases) or recurrent bleeding (one case). Of the 12 scan-positive patients, incorrect localization was noted in two patients due to rapid transit of the labeled RBC in the small bowel. False localization could be prevented by shortening the sequential imaging interval. It is concluded that an RBC scan is a very sensitive and safe tool for detection of GI bleeding of an intermittent nature, because the bleeder can be monitored for 24 hours after a single injection. It can be used as a preangiographic screening test and to guide the surgeon in surgical planning or decision-making.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1352337

  4. Parametric analysis of colony morphology of non-labelled live human pluripotent stem cells for cell quality control

    PubMed Central

    Kato, Ryuji; Matsumoto, Megumi; Sasaki, Hiroto; Joto, Risako; Okada, Mai; Ikeda, Yurika; Kanie, Kei; Suga, Mika; Kinehara, Masaki; Yanagihara, Kana; Liu, Yujung; Uchio-Yamada, Kozue; Fukuda, Takayuki; Kii, Hiroaki; Uozumi, Takayuki; Honda, Hiroyuki; Kiyota, Yasujiro; Furue, Miho K

    2016-01-01

    Given the difficulties inherent in maintaining human pluripotent stem cells (hPSCs) in a healthy state, hPSCs should be routinely characterized using several established standard criteria during expansion for research or therapeutic purposes. hPSC colony morphology is typically considered an important criterion, but it is not evaluated quantitatively. Thus, we designed an unbiased method to evaluate hPSC colony morphology. This method involves a combination of automated non-labelled live-cell imaging and the implementation of morphological colony analysis algorithms with multiple parameters. To validate the utility of the quantitative evaluation method, a parent cell line exhibiting typical embryonic stem cell (ESC)-like morphology and an aberrant hPSC subclone demonstrating unusual colony morphology were used as models. According to statistical colony classification based on morphological parameters, colonies containing readily discernible areas of differentiation constituted a major classification cluster and were distinguishable from typical ESC-like colonies; similar results were obtained via classification based on global gene expression profiles. Thus, the morphological features of hPSC colonies are closely associated with cellular characteristics. Our quantitative evaluation method provides a biological definition of ‘hPSC colony morphology’, permits the non-invasive monitoring of hPSC conditions and is particularly useful for detecting variations in hPSC heterogeneity. PMID:27667091

  5. A reliable indirect cell-labelling protocol for optical imaging allows ex vivo visualisation of mesenchymal stem cells after transplantation.

    PubMed

    Diana, Valentina; Libani, Ilaria Vittoria; Armentero, Marie-Therese; Blandini, Fabio; Lucignani, Giovanni; Silani, Vincenzo; Cova, Lidia; Ottobrini, Luisa

    2013-09-01

    We set out to assess the feasibility of exploiting expression of the mCherry gene, after lentiviral infection, in order visualise bone marrow-derived human mesenchymal stem cells (hMSCs) by optical imaging, and to provide proof of principle of this approach as a method for cell tracking and quantification in pre-clinical models. Commercial hMSCs were infected with a lentiviral vector carrying the mCherry gene under the control of the phosphoglycerate kinase promoter. After extensive in vitro culture, infected hMSCs were analysed for viability, morphology, differentiation capability, and maintenance of fluorescence. Thereafter, mCherry-positive cells were transplanted into unilaterally 6-hydroxy-dopamine lesioned rats (an experimental model of Parkinson's disease). Our analysis showed that hMSCs can be efficiently transduced with the lentiviral vector, retaining their biological features even in the long term. Intrastriatally transplanted mCherry-positive hMSCs can be detected ex vivo by a sensitive cooled CCD camera, both in the whole brain and in serial slices, and relatively quantified. Our protocol was found to be a reliable means of studying the viability of implanted hMSCs. mCherry labelling appears to be readily applicable in the post-transplantation tracking of stem cells and could favour the rapid development of new therapeutic targets for clinical treatments.

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

    PubMed

    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.

  7. Noninvasive Optical Imaging and In Vivo Cell Tracking of Indocyanine Green Labeled Human Stem Cells Transplanted at Superficial or In-Depth Tissue of SCID Mice.

    PubMed

    Sabapathy, Vikram; Mentam, Jyothsna; Jacob, Paul Mazhuvanchary; Kumar, Sanjay

    2015-01-01

    Stem cell based therapies hold great promise for the treatment of human diseases; however results from several recent clinical studies have not shown a level of efficacy required for their use as a first-line therapy, because more often in these studies fate of the transplanted cells is unknown. Thus monitoring the real-time fate of in vivo transplanted cells is essential to validate the full potential of stem cells based therapy. Recent studies have shown how real-time in vivo molecular imaging has helped in identifying hurdles towards clinical translation and designing potential strategies that may contribute to successful transplantation of stem cells and improved outcomes. At present, there are no cost effective and efficient labeling techniques for tracking the cells under in vivo conditions. Indocyanine green (ICG) is a safer, economical, and superior labelling technique for in vivo optical imaging. ICG is a FDA-approved agent and decades of usage have clearly established the effectiveness of ICG for human clinical applications. In this study, we have optimized the ICG labelling conditions that is optimal for noninvasive optical imaging and demonstrated that ICG labelled cells can be successfully used for in vivo cell tracking applications in SCID mice injury models.

  8. Intrinsic Indicator of Photodamage during Label-Free Multiphoton Microscopy of Cells and Tissues

    PubMed Central

    Andresen, Elisabeth F.; Geiger, Kathrin D.; Koch, Edmund; Schackert, Gabriele; Steiner, Gerald; Kirsch, Matthias

    2014-01-01

    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. PMID:25343251

  9. Label-free Screening of Multiple Cell-surface Antigens Using a Single Pore

    NASA Astrophysics Data System (ADS)

    Balakrishnan, Karthik; Chapman, Matthew; Kesavaraju, Anand; Sohn, Lydia

    2012-02-01

    Microfluidic pores have emerged as versatile tools for performing highly sensitive measurements. Pore functionalization can result in slower particle transit rates, thereby providing insight into the properties of particles that travel through a pore. While enhancing utility, functionalizing with only one species limits the broader applicability of pores for biosensing by restricting the insight gained in a single run. We have developed a method of using variable cross-section pores to create unique electronic signatures for reliable detection and automated data analysis. By defining a single pore into sections using common lithography techniques, we can detect when a cell passes through a given pore segment using resistive-pulse sensing. This offers such advantages as 1) the ability to functionalize each portion of a pore with a different antibody that corresponds to different cell surface receptors, enabling label-free multianalyte detection in a single run; and 2) a unique electronic signature that allows for both an accelerated real-time analysis and an additional level of precision to testing. This is particularly critical for clinical diagnostics where accuracy and reliability of results are crucial for healthcare professionals upon which to act.

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

    PubMed

    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.

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

    PubMed Central

    McGuire, Ryan M.; Silberg, Jonathan J.; Pereira, Fred A.; Raphael, Robert M.

    2011-01-01

    Prestin, a multipass transmembrane protein whose N- an 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. PMID:21651892

  12. Tracking of mesenchymal stem cells labeled with gadolinium diethylenetriamine pentaacetic acid by 7T magnetic resonance imaging in a model of cerebral ischemia

    PubMed Central

    GENG, KUAN; YANG, ZHONG XIAN; HUANG, DEXIAO; YI, MEIZI; JIA, YANLONG; YAN, GEN; CHENG, XIAOFANG; WU, RENHUA

    2015-01-01

    Progress in the development of stem cell and gene therapy requires repeatable and non-invasive techniques to monitor the survival and integration of stem cells in vivo with a high temporal and spatial resolution. The purpose of the present study was to examine the feasibility of using the standard contrast agent gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA) to label rat mesenchymal stem cells (MSCs) for stem cell tracking. MSCs, obtained from the bilateral femora of rats, were cultured and propagated. The non-liposomal lipid transfection reagent effectene was then used to induce the intracellular uptake of Gd-DTPA. Electron microscopy was used to detect the distribution of Gd-DTPA particles in the MSCs. The labeling efficiency of the Gd-DTPA particles in the MSCs was determined using spectrophotometry, and MTT and trypan blue exclusion assays were used to evaluate the viability and proliferation of the labeled MSCs. T1-weighted magnetic resonance imaging (MRI) was used to observe the labeled cells in vitro and in the rat brain. Gd-DTPA particles were detected inside the MSCs using transmission electron microscopy and a high labeling efficiency was observed. No difference was observed in cell viability or proliferation between the labeled and unlabeled MSCs (P>0.05). In the in vitro T1-weighted MRI and in the rat brain, a high signal intensity was observed in the labeled MSCs. The T1-weighted imaging of the labeled cells revealed a significantly higher signal intensity compared with that of the unlabeled cells (P<0.05) and the T1 values were significantly lower. The function of the labeled MSCs demonstrated no change following Gd-DTPA labeling, with no evident adverse effect on cell viability or proliferation. Therefore, a change in MR signal intensity was detected in vitro and in vivo, suggesting Gd-DTPA can be used to label MSCs for MRI tracking. PMID:25352164

  13. Label-free in situ imaging of lignification in plant cell walls.

    PubMed

    Schmidt, Martin; Perera, Pradeep; Schwartzberg, Adam M; Adams, Paul D; Schuck, P James

    2010-11-01

    Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find cost-effective and sustainable solutions has become a topical and critical task. Lignocellulosic biomass is poised to become the primary source of biomass for the conversion to liquid biofuels. However, the recalcitrance of these plant cell wall materials to cost-effective and efficient degradation presents a major impediment for their use in the production of biofuels and chemicals. In particular, lignin, a complex and irregular poly-phenylpropanoid heteropolymer, becomes problematic to the postharvest deconstruction of lignocellulosic biomass. For example in biomass conversion for biofuels, it inhibits saccharification in processes aimed at producing simple sugars for fermentation. The effective use of plant biomass for industrial purposes is in fact largely dependent on the extent to which the plant cell wall is lignified. The removal of lignin is a costly and limiting factor and lignin has therefore become a key plant breeding and genetic engineering target in order to improve cell wall conversion. Analytical tools that permit the accurate rapid characterization of lignification of plant cell walls become increasingly important for evaluating a large number of breeding populations. Extractive procedures for the isolation of native components such as lignin are inevitably destructive, bringing about significant chemical and structural modifications. Analytical chemical in situ methods are thus invaluable tools for the compositional and structural characterization of lignocellulosic materials. Raman microscopy is a technique that relies on inelastic or Raman scattering of monochromatic light, like that from a laser, where the shift in energy of the laser photons is related to molecular vibrations and presents an intrinsic label-free molecular "fingerprint" of the sample. Raman microscopy can afford non

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

    PubMed

    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.

  15. Design of an automated algorithm for labeling cardiac blood pool in gated SPECT images of radiolabeled red blood cells

    SciTech Connect

    Hebert, T.J. |; Moore, W.H.; Dhekne, R.D.; Ford, P.V.; Wendt, J.A.; Murphy, P.H.; Ting, Y.

    1996-08-01

    The design of an automated computer algorithm for labeling the cardiac blood pool within gated 3-D reconstructions of the radiolabeled red blood cells is investigated. Due to patient functional abnormalities, limited resolution, and noise, certain spatial and temporal features of the cardiac blood pool that one would anticipate finding in every study are not present in certain frames or with certain patients. The labeling of the cardiac blood pool requires an algorithm that only relies upon features present in all patients. The authors investigate the design of a fully-automated region growing algorithm for this purpose.

  16. Label-Free Detection of Rare Cell in Human Blood Using Gold Nano Slit Surface Plasmon Resonance

    PubMed Central

    Mousavi, Mansoureh Z.; Chen, Huai-Yi; Hou, Hsien-San; Chang, Chou-Yuan-Yuan; Roffler, Steve; Wei, Pei-Kuen; Cheng, Ji-Yen

    2015-01-01

    Label-free detection of rare cells in biological samples is an important and highly demanded task for clinical applications and various fields of research, such as detection of circulating tumor cells for cancer therapy and stem cells studies. Surface Plasmon Resonance (SPR) as a label-free method is a promising technology for detection of rare cells for diagnosis or research applications. Short detection depth of SPR (400 nm) provides a sensitive method with minimum interference of non-targets in the biological samples. In this work, we developed a novel microfluidic chip integrated with gold nanoslit SPR platform for highly efficient immunomagnetic capturing and detection of rare cells in human blood. Our method offers simple yet efficient detection of target cells with high purity. The approach for detection consists of two steps. Target cells are firs captured on functionalized magnetic nanoparticles (MNPs) with specific antibody I. The suspension containing the captured cells (MNPs-cells) is then introduced into a microfluidic chip integrated with a gold nanoslit film. MNPs-cells bind with the second specific antibody immobilized on the surface of the gold nanoslit and are therefore captured on the sensor active area. The cell binding on the gold nanoslit was monitored by the wavelength shift of the SPR spectrum generated by the gold nanoslits. PMID:25806834

  17. Relaxation effects of Ferucarbotran-labeled mesenchymal stem cells at 1.5T and 3T: Discrimination of viable from lysed cells

    PubMed Central

    Henning, Tobias D; Wendland, Michael F; Golovko, Daniel; Sutton, Elizabeth J; Sennino, Barbara; Malek, Farbod; Bauer, Jan S; McDonald, Donald M; Daldrup-Link, Heike

    2010-01-01

    Human mesenchymal stem cells (hMSC) were labeled with Ferucarbotran by simple incubation and cultured for up to 14 days. Iron content was determined by spectrometry and the intracellular localization of the contrast agent uptake was studied by electron and confocal microscopy. At various time points after labeling, reaching from 1 to 14 days, samples with viable or lysed labeled hMSCs, as well as non-labeled controls underwent MR imaging. SE- and GE-sequences with multiple TRs and TEs were used at 1.5T and 3T on a clinical scanner. Spectrometry showed an initial iron oxide uptake of 7.08 pg per cell. Microscopy studies revealed lysosomal compartmentalization. Contrast agent effects of hMSC were persistent for up to 14 days after labeling. A marked difference in the T2-effect of compartmentalized iron oxides compared to free iron oxides was found on T2-weighted sequences, but not on T2*-sequences. The observed differences may be explained by the loss of compartmentalization of iron oxide particles, the uniformity of distribution and the subsequent increase in dephasing of protons on SE images. These results show that viable cells with compartmentalized iron oxides may – in principle – be distinguished from lysed cells or released iron oxides. PMID:19353670

  18. Separation of SSEA-4 and TRA-1-60 labelled undifferentiated human embryonic stem cells from a heterogeneous cell population using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS).

    PubMed

    Fong, Chui Yee; Peh, Gary S L; Gauthaman, Kalamegam; Bongso, Ariff

    2009-03-01

    A major concern in human embryonic stem cell (hESC)-derived cell replacement therapy is the risk of tumorigenesis from undifferentiated hESCs residing in the population of hESC-derived cells. Separation of these undifferentiated hESCs from the differentiated derivatives using cell sorting methods may be a plausible approach in overcoming this problem. We therefore explored magnetic activated cell sorting (MACS) and fluorescence activated cell sorting (FACS) to separate labelled undifferentiated hESCs from a heterogeneous population of hESCs and hepatocellular carcinoma cells (HepG2) deliberately mixed respectively at different ratios (10:90, 20:80, 30:70, 40:60 and 50:50) to mimic a standard in vitro differentiation protocol, instead of using a hESC-differentiated cell population, so that we could be sure of the actual number of cells separated. HES-3 and HES-4 cells were labelled in separate experiments for the stem cell markers SSEA-4 and TRA-1-60 using primary antibodies. Anti-PE magnetic microbeads that recognize the PE-conjugated SSEA-4 labelled hESCs was added to the heterogeneous cell mixture and passed through the MACS column. The cells that passed through the column ('flow-through' fraction) and those retained ('labelled' fraction') were subsequently analysed using FACS. The maximum efficacy of hESCs retention using MACS was 81.0 +/- 2.9% (HES-3) and 83.6 +/- 4.2% (HES-4). Using FACS, all the undifferentiated hESCs labelled with the two cell-surface markers could be removed by selective gating. Both hESCs and HepG2 cells in the 'flow-through' fraction following MACS separation were viable in culture whereas by FACS separation only the HepG2 cells were viable. FACS efficiently helps to eliminate the undifferentiated hESCs based on their cell-surface antigens expressed.

  19. High Cell Density Production of Europium-Labeled Escherichia coli for Tracing of Bacteria in Mantled Karst of Northwest Arkansas

    NASA Astrophysics Data System (ADS)

    Ting, T.; Thoma, G. J.; Beitle, R. B.; Davis, R. K.; Brahana, J. V.; Liu, H.

    2004-12-01

    The preparation of europium-labeled E. coli as a bacterial tracer in our study is separated into two major steps: the production of large quantities of cells, and the labeling of the cells at high density. Indigenous E. coli isolated from a natural spring at the University of Arkansas's Savoy Experimental Watershed (SEW), Savoy, Arkansas was fermented in BIOFLO II (New Brunswick Scientific, Edison, NJ) bioreactor using a fed-batch technique. Either a concentrated glucose solution or an ammonium hydroxide solution was pulsed into the reactor automatically using closed-loop pH control in a reactor feeding strategy designed to optimize cell growth. E. coli cells were harvested at the stationary phase of the bacterial growth profile, washed and centrifuged prior to the europium labeling step. A concentrated europium chloride solution was prepared by dissolving europium (III) chloride in 1-L of deionized water; the salt solution was chilled at 6oC overnight. A batch of 100-g wet weight of the washed E. coli was suspended in the chilled europium salt solution, and the cells were incubated at 6oC for 2 hours with stirring. After the cold incubation, the cells were washed with chilled deionized water and centrifuged repeatedly to remove excess europium. We have successfully prepared 760-g wet weight of labeled E. coli using the high cell density fermentation and europium labeling technique in a 9-day period. Preparation of large quantities of viable europium-tagged bacteria is critical for use as an environmental tracer. The europium uptake by the E. coli was found to be 15-mg europium per gram of labeled cell (wet weight). A field injection of multiple tracers along with the europium-tagged E. coli was successfully performed during the summer of 2004 at SEW to elucidate the transport, storage and viability of fecal contaminants in a karst basin. Prior investigations suggest that, unlike conservative tracers, E. coli become deposited along the flow path in the aquifer, and

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

    PubMed Central

    2005-01-01

    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. PMID:16192680

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

    PubMed

    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

  2. Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis

    PubMed Central

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

    2013-01-01

    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

  3. Metabolic Labeling of Caenorhabditis elegans Primary Embryonic Cells with Azido-Sugars as a Tool for Glycoprotein Discovery

    PubMed Central

    Burnham-Marusich, Amanda R.; Snodgrass, Casey J.; Johnson, Anna M.; Kiyoshi, Conrad M.; Buzby, Sarah E.; Gruner, Matt R.; Berninsone, Patricia M.

    2012-01-01

    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. PMID:23152843

  4. Investigation of Adaptive Responses in Bystander Cells in 3D Cultures Containing Tritium-Labeled and Unlabeled Normal Human Fibroblasts

    PubMed Central

    Pinto, Massimo; Azzam, Edouard I.; Howell, Roger W.

    2010-01-01

    The study of radiation-induced bystander effects in normal human cells maintained in three-dimensional (3D) architecture provides more in vivo-like conditions and is relevant to human risk assessment. Linear energy transfer, dose and dose rate have been considered as critical factors in propagating radiation-induced effects. This investigation uses an in vitro 3D tissue culture model in which normal AG1522 human fibroblasts are grown in a carbon scaffold to investigate induction of a G1 arrest in bystander cells that neighbor radiolabeled cells. Cell cultures were co-pulse-labeled with [3H]deoxycytidine (3HdC) to selectively irradiate a minor fraction of cells with 1–5 keV/μm β particles and bromodeoxyuridine (BrdU) to identify the radiolabeled cells using immunofluorescence. The induction of a G1 arrest was measured specifically in unlabeled cells (i.e. bystander cells) using a flow cytometry-based version of the cumulative labeling index assay. To investigate the relationship between bystander effects and adaptive responses, cells were challenged with an acute 4 Gy γ-radiation dose after they had been kept under the bystander conditions described above for several hours, and the regulation of the radiation-induced G1 arrest was measured selectively in bystander cells. When the average dose rate in 3HdC-labeled cells (<16% of population) was 0.04–0.37 Gy/h (average accumulated dose 0.14–10 Gy), no statistically significant stressful bystander effects or adaptive bystander effects were observed as measured by magnitude of the G1 arrest, micronucleus formation, or changes in mitochondrial membrane potential. Higher dose rates and/or higher LET may be required to observe stressful bystander effects in this experimental system, whereas lower dose rates and challenge doses may be required to detect adaptive bystander responses. PMID:20681788

  5. Label-free selection and enrichment of liver cancer stem cells by surface niches build up with polyelectrolyte multilayer films.

    PubMed

    Lee, I-Chi; Chang, Jen-Fu

    2015-01-01

    Recent studies indicate that a small population of cancer cells exhibits stem cell properties and are referred to as cancer-initiating or cancer stem cells (CSCs). The selection and identification of cancer stem cells through methods require well-defined biomarkers and immunolabeling procedures are complicated and often unreliable. Herein, we fabricated a series of microenviroment by using polyelectrolyte multilayers (PEM) nanofilms to program and mimic hepatocellular carcinoma CSCs niches for CSCs selection with a label-free method. When cultured on PEM substrates, human cancer cell lines-Huh7 cells grew into individual round colonies and these cells displayed high marker expression of CSCs. Especially, these selected cells demonstrated significant chemo-resistant property in comparison with normal population. Therefore, we believed that niches selection and colony formation method may provide a new strategy on CSCs selection and drug evaluation for cancer therapy. PMID:25461919

  6. BODIPY-labeled DC-SIGN-targeting glycodendrons efficiently internalize and route to lysosomes in human dendritic cells.

    PubMed

    Ribeiro-Viana, Renato; García-Vallejo, Juan J; Collado, Daniel; Pérez-Inestrosa, Ezequiel; Bloem, Karien; van Kooyk, Yvette; Rojo, Javier

    2012-10-01

    Glycodendrons bearing nine copies of mannoses or fucoses have been prepared by an efficient convergent strategy based on Cu(I) catalyzed azide-alkyne cycloaddition (CuAAC). These glycodendrons present a well-defined structure and have an adequate size and shape to interact efficiently with the C-type lectin DC-SIGN. We have selected a BODIPY derivative to label these glycodendrons due to its interesting physical and chemical properties as chromophore. These BODIPY-labeled glycodendrons were internalized into dendritic cells by mean of DC-SIGN. The internalized mannosylated and fucosylated dendrons are colocalized with LAMP1, which suggests routing to lysosomes. The interaction of these glycodendrons with DC-SIGN at the surface of dendritic cells did not induce maturation of the cells. Signaling analysis by checking different cytokines indicated also the lack of induction the expression of inflammatory and noninflammatory cytokines by these second generation glycodendrons. PMID:22920925

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

    NASA Astrophysics Data System (ADS)

    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.

  8. Umbilical cord mesenchymal stem cells labeled with multimodal iron oxide nanoparticles with fluorescent and magnetic properties: application for in vivo cell tracking

    PubMed Central

    Sibov, Tatiana T; Pavon, Lorena F; Miyaki, Liza A; Mamani, Javier B; Nucci, Leopoldo P; Alvarim, Larissa T; Silveira, Paulo H; Marti, Luciana C; Gamarra, LF

    2014-01-01

    Here we describe multimodal iron oxide nanoparticles conjugated to Rhodamine-B (MION-Rh), their stability in culture medium, and subsequent validation of an in vitro protocol to label mesenchymal stem cells from umbilical cord blood (UC-MSC) with MION-Rh. These cells showed robust labeling in vitro without impairment of their functional properties, the viability of which were evaluated by proliferation kinetic and ultrastructural analyzes. Thus, labeled cells were infused into striatum of adult male rats of animal model that mimic late onset of Parkinson’s disease and, after 15 days, it was observed that cells migrated along the medial forebrain bundle to the substantia nigra as hypointense spots in T2 magnetic resonance imaging. These data were supported by short-term magnetic resonance imaging. Studies were performed in vivo, which showed that about 5 × 105 cells could be efficiently detected in the short term following infusion. Our results indicate that these labeled cells can be efficiently tracked in a neurodegenerative disease model. PMID:24531365

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

    PubMed

    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

  10. Detection of metastatic tumour cells in routine bone marrow smears by immuno-alkaline phosphatase labelling with monoclonal antibodies.

    PubMed

    Ghosh, A K; Erber, W N; Hatton, C S; O'Connor, N T; Falini, B; Osborn, M; Mason, D Y

    1985-09-01

    The present study describes 11 cases (10 carcinomas, one rhabdomyosarcoma) in which immuno-alkaline phosphatase labelling with monoclonal antibodies was used to demonstrate metastatic cells in routine smears of aspirated bone marrow. Carcinoma cells were detected using antibodies against epithelial cytokeratins, milk fat globule membrane antigen and carcinoembryonic antigen, and rhabdomyosarcoma cells with monoclonal anti-desmin. In four of the carcinoma cases it had not been possible to identify malignant cells in routinely stained marrow smears, whilst the case of disseminated rhabdomyosarcoma had initially been diagnosed (and treated) as a case of acute lymphoblastic leukaemia. The anti-cytokeratin antibody was found to be the most valuable of the anti-epithelial reagents used, since it labelled malignant cells in all of the 10 cases of carcinoma and gave the strongest reactions. These results suggest that immunocytochemical labelling should be used in cases of suspected carcinoma whenever conventional examination of marrow smears yields negative results, and furthermore (as illustrated by the case of rhabdomyosarcoma) that the technique is of value for identifying the true nature of poorly differentiated neoplasms in bone marrow.

  11. The study of optimal condition of SPIO labeling human lung adenocarcinoma cell line (SPC-A-1)

    NASA Astrophysics Data System (ADS)

    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.

  12. 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.

    PubMed

    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.

  13. Assessment of soft tissue hemangiomas in children utilizing Tc-99m labelled red blood cells

    SciTech Connect

    Miller, J.H.

    1984-01-01

    Hemangiomas may present in infancy as soft tissue masses. Occasionally these lesions may be extensive or may not be clinically recognized as a hemangioma, often causing concern for the presence of a malignant lesion. In later childhood these lesions, which may be occult, may cause overgrowth of an extremity. Evaluation of soft tissue masses suspected of being a hemangioma utilizing Technetium 99m labelled red blood cells has been very valuable. This method allows a dynamic evaluation of first pass blood flow. Subsequent static scintiphotos allow an assessment of the lesion itself. These scintiphotos may be obtained sequentially to evaluate therapy. Twenty patients were evaluated by this method ranging in age from two months to eleven years. There were 13 females and seven males. Lesions evaluated by this method include six hemangiomas of the head and neck: parotic region (2), facial (3), and tongue (1). Extremity lesions were evaluated in six children including both upper extremity (1) and lower extremity (5). Torso lesions evaluated include chest wall (2), abdominal wall (2), and one hemangioma of the gut. This procedure is quickly performed on an outpatient basis, has high anatomic resolution, provides and assessment of these lesions in a manner not available by any other imaging procedure and usually requires no sedation. The radiation exposure for this procedure is low (approximately, a 400mR total body dose) and has been well tolerated by both patients and their parents. Scintigraphic evaluation should be the first diagnostic method utilized in the evaluation of these lesions.

  14. Sickle cell anemia: reference values of cerebral blood flow determined by continuous arterial spin labeling MRI.

    PubMed

    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.

  15. A novel self-powered and sensitive label-free DNA biosensor in microbial fuel cell.

    PubMed

    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

  16. Labeling of Chromosomes in Cell Development and the Appearance of Monozygotic Twins

    PubMed Central

    Jim, Carol; Berkovich, Simon

    2015-01-01

    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 (1/2)K, and we discover that the probability of monozygotic quadruplets, or triplets as in the case of the death of an embryo, is (1/8)K, 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. PMID:26185760

  17. Measurement of Retinal Blood Flow Using Fluorescently Labeled Red Blood Cells1,2,3

    PubMed Central

    Kornfield, Tess E.

    2015-01-01

    Abstract Blood flow is a useful indicator of the metabolic state of the retina. However, accurate measurement of retinal blood flow is difficult to achieve in practice. Most existing optical techniques used for measuring blood flow require complex assumptions and calculations. We describe here a simple and direct method for calculating absolute blood flow in vessels of all sizes in the rat retina. The method relies on ultrafast confocal line scans to track the passage of fluorescently labeled red blood cells (fRBCs). The accuracy of the blood flow measurements was verified by (1) comparing blood flow calculated independently using either flux or velocity combined with diameter measurements, (2) measuring total retinal blood flow in arterioles and venules, (3) measuring blood flow at vessel branch points, and (4) measuring changes in blood flow in response to hyperoxic and hypercapnic challenge. Confocal line scans oriented parallel and diagonal to vessels were used to compute fRBC velocity and to examine velocity profiles across the width of vessels. We demonstrate that these methods provide accurate measures of absolute blood flow and velocity in retinal vessels of all sizes. PMID:26082942

  18. Autologous Bone Marrow Mononuclear Cell Therapy for Autism: An Open Label Proof of Concept Study

    PubMed Central

    Sharma, Alok; Gokulchandran, Nandini; Sane, Hemangi; Nagrajan, Anjana; Kulkarni, Pooja; Shetty, Akshata; Mishra, Priti; Kali, Mrudula; Biju, Hema; Badhe, Prerna

    2013-01-01

    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. PMID:24062774

  19. High-Throughput Single-Cell Labeling (Hi-SCL) for RNA-Seq Using Drop-Based Microfluidics.

    PubMed

    Rotem, Assaf; Ram, Oren; Shoresh, Noam; Sperling, Ralph A; Schnall-Levin, Michael; Zhang, Huidan; Basu, Anindita; Bernstein, Bradley E; Weitz, David A

    2015-01-01

    The importance of single-cell level data is increasingly appreciated, and significant advances in this direction have been made in recent years. Common to these technologies is the need to physically segregate individual cells into containers, such as wells or chambers of a micro-fluidics chip. High-throughput Single-Cell Labeling (Hi-SCL) in drops is a novel method that uses drop-based libraries of oligonucleotide barcodes to index individual cells in a population. The use of drops as containers, and a microfluidics platform to manipulate them en-masse, yields a highly scalable methodological framework. Once tagged, labeled molecules from different cells may be mixed without losing the cell-of-origin information. Here we demonstrate an application of the method for generating RNA-sequencing data for multiple individual cells within a population. Barcoded oligonucleotides are used to prime cDNA synthesis within drops. Barcoded cDNAs are then combined and subjected to second generation sequencing. The data are deconvoluted based on the barcodes, yielding single-cell mRNA expression data. In a proof-of-concept set of experiments we show that this method yields data comparable to other existing methods, but with unique potential for assaying very large numbers of cells.

  20. High-Throughput Single-Cell Labeling (Hi-SCL) for RNA-Seq Using Drop-Based Microfluidics

    PubMed Central

    Sperling, Ralph A.; Schnall-Levin, Michael; Zhang, Huidan; Basu, Anindita; Bernstein, Bradley E.; Weitz, David A.

    2015-01-01

    The importance of single-cell level data is increasingly appreciated, and significant advances in this direction have been made in recent years. Common to these technologies is the need to physically segregate individual cells into containers, such as wells or chambers of a micro-fluidics chip. High-throughput Single-Cell Labeling (Hi-SCL) in drops is a novel method that uses drop-based libraries of oligonucleotide barcodes to index individual cells in a population. The use of drops as containers, and a microfluidics platform to manipulate them en-masse, yields a highly scalable methodological framework. Once tagged, labeled molecules from different cells may be mixed without losing the cell-of-origin information. Here we demonstrate an application of the method for generating RNA-sequencing data for multiple individual cells within a population. Barcoded oligonucleotides are used to prime cDNA synthesis within drops. Barcoded cDNAs are then combined and subjected to second generation sequencing. The data are deconvoluted based on the barcodes, yielding single-cell mRNA expression data. In a proof-of-concept set of experiments we show that this method yields data comparable to other existing methods, but with unique potential for assaying very large numbers of cells. PMID:26000628

  1. Specific detection of CD133-positive tumor cells with iron oxide nanoparticles labeling using noninvasive molecular magnetic resonance imaging

    PubMed Central

    Chen, Ya-Wen; Liou, Gunn-Guang; Pan, Huay-Ben; Tseng, Hui-Hwa; Hung, Yu-Ting; Chou, Chen-Pin

    2015-01-01

    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 reactive oxygen species production showed no significant differences in tumor cells with or without labeling of USPIO-CD133 Ab. In vivo imaging of CD133-positive cells was demonstrated by intravenous injection of USPIO-CD133 Ab in mice with HT29 xenografted tumors. The MRI of HT29 xenografts showed several clusters of hypotensive regions that correlated with CD133 expression and Prussian blue staining for iron. In rat, brain tumors induced by transplacental ENU mutagenesis, several clusters of hypointensive zones were observed in CD133-expressing brain tumors by MRI and intravenously administered USPIO-CD133 Ab. Conclusion Combination of USPIO-CD133 Ab and MRI is valuable in recognizing CD133-expressing tumor cells in vitro, extracellularly

  2. Whole-brain neural network analysis (connectomics) using cell lineage-based neuron-labeling method.

    PubMed

    Ito, Kei; Ito, Masayoshi

    2014-11-01

    The brain is a computing machine that receives input signals from sensory neurons, calculates best responses to changing environments, and sends output signals to motor muscles. How such computation is materialized remains largely unknown. Understanding the entire wiring network of neural connections in the brain, which is recently called the connectomics (connection + omics), should provide indispensable insights on this problem.To resolve the circuit diagram from the tangled thickets of neural fibers, only a small subset of neurons should be visualized at one time. Previous studies visualized such selective cells by injecting dyes or by detecting specific molecules or gene expression patterns using antibodies and expression driver strains. These approaches were unfortunately not efficient enough for identifying all the brain cells in a comprehensive and systematic manner.Neurons are generated by neural stem cells. The entire neural population can therefore be divided into a finite number of families - or clones - of the cells that are the progeny of each single stem cell. The central brain of the fruit fly Drosophila melanogaster consists of about 15,000 neurons per side and is made by utmost 100 stem cells. By genetically labeling one of such stem cells and tracing the projection patterns of its progeny in the adult brain, we were able to identify the neural projections of almost all the clonal cell groups.To visualize these neural projections, we made serial optical sections of the fly brain using laser confocal microscopy. Because of its relatively small size (0.6-mm wide and less than 0.3-mm thick), the entire fly brain can be imaged using high-resolution objectives with n.a. 1.2. Neuronal fibers are visualized by ectopically expressed cytoplasmic and membrane-bound fluorescent proteins, and the output synaptic sites are visualized with ectopically expressed tag proteins that are fused with the proteins associated with synaptic vesicles. In addition, density

  3. Dual luciferase labelling for non-invasive bioluminescence imaging of mesenchymal stromal cell chondrogenic differentiation in demineralized bone matrix scaffolds.

    PubMed

    Vilalta, Marta; Jorgensen, Christian; Dégano, Irene R; Chernajovsky, Yuti; Gould, David; Noël, Danièle; Andrades, José A; Becerra, José; Rubio, Nuria; Blanco, Jerónimo

    2009-10-01

    Non-invasive bioluminescence imaging (BLI) to monitor changes in gene expression of cells implanted in live animals should facilitate the development of biomaterial scaffolds for tissue regeneration. We show that, in vitro, induction of chondrogenic differentiation in mouse bone marrow stromal cell line (CL1) and human adipose tissue derived mesenchymal stromal cells (hAMSCs), permanently transduced with a procollagen II (COL2A1) promoter driving a firefly luciferase gene reporter (PLuc) (COL2A1p.PLuc), induces PLuc expression in correlation with increases in COL2A1 and Sox9 mRNA expression and acquisition of chondrocytic phenotype. To be able to simultaneously monitor in vivo cell differentiation and proliferation, COL2A1p.PLuc labelled cells were also genetically labelled with a renilla luciferase (RLuc) gene driven by a constitutively active cytomegalovirus promoter, and then seeded in demineralized bone matrix (DBM) subcutaneously implanted in SCID mice. Non-invasive BLI monitoring of the implanted mice showed that the PLuc/RLuc ratio reports on gene expression changes indicative of cell differentiation. Large (CL1) and moderated (hAMSCs) changes in the PLuc/RLuc ratio over a 6 week period, revealed different patterns of in vivo chondrogenic differentiation for the CL1 cell line and primary MSCs, in agreement with in vitro published data and our results from histological analysis of DBM sections. This double bioluminescence labelling strategy together with BLI imaging to analyze behaviour of cells implanted in live animals should facilitate the development of progenitor cell/scaffold combinations for tissue repair.

  4. Shrinkage of X cells in the lateral geniculate nucleus after monocular deprivation revealed by FoxP2 labeling.

    PubMed

    Duffy, Kevin R; Holman, Kaitlyn D; Mitchell, Donald E

    2014-05-01

    The parallel processing of visual features by distinct neuron populations is a central characteristic of the mammalian visual system. In the A laminae of the cat dorsal lateral geniculate nucleus (dLGN), parallel processing streams originate from two principal neuron types, called X and Y cells. Disruption of visual experience early in life by monocular deprivation has been shown to alter the structure and function of Y cells, but the extent to which deprivation influences X cells remains less clear. A transcription factor, FoxP2, has recently been shown to selectively label X cells in the ferret dLGN and thus provides an opportunity to examine whether monocular deprivation alters the soma size of X cells. In this study, FoxP2 labeling was examined in the dLGN of normal and monocularly deprived cats. The characteristics of neurons labeled for FoxP2 were consistent with FoxP2 being a marker for X cells in the cat dLGN. Monocular deprivation for either a short (7 days) or long (7 weeks) duration did not alter the density of FoxP2-positive neurons between nondeprived and deprived dLGN layers. However, for each deprived animal examined, measurement of the cross-sectional area of FoxP2-positive neurons (X cells) revealed that within deprived layers, X cells were smaller by approximately 20% after 7 days of deprivation, and by approximately 28% after 7 weeks of deprivation. The observed alteration to the cross-sectional area of X cells indicates that perturbation of this major pathway contributes to the functional impairments that develop from monocular deprivation.

  5. Filming a live cell by scanning electrochemical microscopy: label-free imaging of the dynamic morphology in real time

    PubMed Central

    2012-01-01

    The morphology of a live cell reflects the organization of the cytoskeleton and the healthy status of the cell. We established a label-free platform for monitoring the changing morphology of live cells in real time based on scanning electrochemical microscopy (SECM). The dynamic morphology of a live human bladder cancer cell (T24) was revealed by time-lapse SECM with dissolved oxygen in the medium solution as the redox mediator. Detailed local movements of cell membrane were presented by time-lapse cross section lines extracted from time-lapse SECM. Vivid dynamic morphology is presented by a movie made of time-lapse SECM images. The morphological change of the T24 cell by non-physiological temperature is in consistence with the morphological feature of early apoptosis. To obtain dynamic cellular morphology with other methods is difficult. The non-invasive nature of SECM combined with high resolution realized filming the movements of live cells. PMID:22436305

  6. Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope

    PubMed Central

    Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

    2015-01-01

    Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications. PMID:26525841

  7. Isolation of Plant Nuclei at Defined Cell Cycle Stages Using EdU Labeling and Flow Cytometry.

    PubMed

    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

  8. Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope

    NASA Astrophysics Data System (ADS)

    Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

    2015-11-01

    Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications.

  9. Isolation of Plant Nuclei at Defined Cell Cycle Stages Using EdU Labeling and Flow Cytometry.

    PubMed

    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.

  10. Human amniotic fluid stem cells labeled with up-conversion nanoparticles for imaging-monitored repairing of acute lung injury.

    PubMed

    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

  11. Label-free 3D refractive-index acquisition by micro-manipulations of cells in suspension (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Shaked, Natan T.

    2016-03-01

    Our latest methods for non-invasive label-free acquisition of the three-dimensional (3-D) refractive-index maps of live cells in suspension are reviewed. These methods are based on the acquisition of off-axis interferograms of single or multiple cells in suspension from different angles using an external interferometric module, while fully rotating each cell using micro-manipulations. The interferometric projections are processed via computed tomographic phase microscopy reconstruction technique, which considers optical diffraction effects, into the 3-D refractive-index structure of the suspended cell. Till now, tomographic phase microscopy was obtained by acquiring a series of interferograms of the light transmitted through the sample in different angles by either using an entire sample rotation, or patch clamping a single cell, which is invasive to the cells, or alternatively, using various angles of illumination, which causes a limited acceptance angle, and an incomplete 3-D Fourier spectrum. In contrast, our methods allow fast acquisition with full angular range, and thus obtain an accurate 3-D refractive-index map of the imaged cell. By inspection of the 3-D refractive-index distribution of cells in suspension, the proposed methods can be useful for high-throughput, label-free characterization of biological processes and cellular transformations from healthy to pathological conditions.

  12. Rare cell proteomic reactor applied to stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics study of human embryonic stem cell differentiation.

    PubMed

    Tian, Ruijun; Wang, Shuai; Elisma, Fred; Li, Li; Zhou, Hu; Wang, Lisheng; Figeys, Daniel

    2011-02-01

    The molecular basis governing the differentiation of human embryonic stem cells (hESCs) remains largely unknown. Systems-level analysis by proteomics provides a unique approach to tackle this question. However, the requirement of a large number of cells for proteomics analysis (i.e. 10(6)-10(7) cells) makes this assay challenging, especially for the study of rare events during hESCs lineage specification. Here, a fully integrated proteomics sample processing and analysis platform, termed rare cell proteomic reactor (RCPR), was developed for large scale quantitative proteomics analysis of hESCs with ∼50,000 cells. hESCs were completely extracted by a defined lysis buffer, and all of the proteomics sample processing procedures, including protein preconcentration, reduction, alkylation, and digestion, were integrated into one single capillary column with a strong cation exchange monolith matrix. Furthermore, on-line two-dimensional LC-MS/MS analysis was performed directly using RCPR as the first dimension strong cation exchange column. 2,281 unique proteins were identified on this system using only 50,000 hESCs. For stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative study, a ready-to-use and chemically defined medium and an in situ differentiation procedure were developed for complete SILAC labeling of hESCs with well characterized self-renewal and differentiation properties. Mesoderm-enriched differentiation was studied by RCPR using 50,000 hESCs, and 1,086 proteins were quantified with a minimum of two peptides per protein. Of these, 56 proteins exhibited significant changes during mesoderm-enriched differentiation, and eight proteins were demonstrated for the first time to be overexpressed during early mesoderm development. This work provides a new platform for the study of rare cells and in particular for further elucidating proteins that govern the mesoderm lineage specification of human pluripotent stem cells.

  13. Differentiation of MCF-7 tumor cells from leukocytes and fibroblast cells using epithelial cell adhesion molecule targeted multicore surface-enhanced Raman spectroscopy labels

    NASA Astrophysics Data System (ADS)

    Freitag, Isabel; Matthäus, Christian; Csaki, Andrea; Clement, Joachim H.; Cialla-May, Dana; Weber, Karina; Krafft, Christoph; Popp, Jürgen

    2015-05-01

    Identification of tumor and normal cells is a promising application of Raman spectroscopy. The throughput of Raman-assisted cell sorting is limited by low sensitivity. Surface-enhanced Raman spectroscopy (SERS) is a well-recognized candidate to increase the intensity of Raman signals of cells. First, different strategies are summarized to detect tumor cells using targeted SERS probes. Then, a protocol is described to prepare multicore-SERS-labels (MSLs) by aggregating gold nanoparticles, coating with a reporter molecule and a thin silver shell to further boost enhancement, encapsulating with a stable silica layer, and functionalizing by epithelial cell adhesion molecule (EpCAM) antibodies. Raman, dark field and fluorescence microscopy proved the specific and nonspecific binding of functionalized and nonfunctionalized MSLs to MCF-7 tumor cells, leukocytes from blood, and nontransformed human foreskin fibroblasts. Raman imaging and dark field microscopy indicated no uptake of MSLs, yet binding to the cellular membrane. Viability tests were performed with living tumor cells to demonstrate the low toxicity of MSL-EpCAM. The SERS signatures were detected from cells with exposure times down to 25 ms at 785-nm laser excitation. The prospects of these MSLs in multiplex assays, for enumeration and sorting of circulating tumor cells in microfluidic chips, are discussed.

  14. Improved biocompatibility and efficient labeling of neural stem cells with poly(L-lysine)-coated maghemite nanoparticles

    PubMed Central

    Ahmed, Lada Brkić; Babič, Michal; Šlouf, Miroslav; Horák, Daniel

    2016-01-01

    Summary Background: Cell tracking is a powerful tool to understand cellular migration, dynamics, homing and function of stem cell transplants. Nanoparticles represent possible stem cell tracers, but they differ in cellular uptake and side effects. Their properties can be modified by coating with different biocompatible polymers. To test if a coating polymer, poly(L-lysine), can improve the biocompatibility of nanoparticles applied to neural stem cells, poly(L-lysine)-coated maghemite nanoparticles were prepared and characterized. We evaluated their cellular uptake, the mechanism of internalization, cytotoxicity, viability and proliferation of neural stem cells, and compared them to the commercially available dextran-coated nanomag®-D-spio nanoparticles. Results: Light microscopy of Prussian blue staining revealed a concentration-dependent intracellular uptake of iron oxide in neural stem cells. The methyl thiazolyl tetrazolium assay and the calcein acetoxymethyl ester/propidium iodide assay demonstrated that poly(L-lysine)-coated maghemite nanoparticles scored better than nanomag®-D-spio in cell labeling efficiency, viability and proliferation of neural stem cells. Cytochalasine D blocked the cellular uptake of nanoparticles indicating an actin-dependent process, such as macropinocytosis, to be the internalization mechanism for both nanoparticle types. Finally, immunocytochemistry analysis of neural stem cells after treatment with poly(L-lysine)-coated maghemite and nanomag®-D-spio nanoparticles showed that they preserve their identity as neural stem cells and their potential to differentiate into all three major neural cell types (neurons, astrocytes and oligodendrocytes). Conclusion: Improved biocompatibility and efficient cell labeling makes poly(L-lysine)-coated maghemite nanoparticles appropriate candidates for future neural stem cell in vivo tracking studies. PMID:27547609

  15. A low-toxic artificial fluorescent glycoprotein can serve as an efficient cytoplasmic labeling in living cell.

    PubMed

    Si, Jiangju; Liang, Dawei; Kong, Dan; Wu, Sufang; Yuan, Lan; Xiang, Yan; Jiang, Lei

    2015-03-01

    To maintain the virtue of good optical property and discard the dross of conventional fluorescent staining dyes, we provide a strategy for designing new fluorescent scaffolds. In this study, a novel fluorescent labeling glycoprotein (chitosan-poly-L-cysteine, CPC) was synthesized through graft copolymerization. CPC gives emission peak at 465-470 nm when excited at 386 nm. The submicro-scale CPC microspheres could be localized and persisted specifically in the cytoplasm of living cells, with strong blue fluorescence. Moreover, CPC was highly resistant to photo bleaching, the fluorescence was remained stable for up to 72 h as the cells grew and developed. The glycoprotein CPC was bio-compatible and in zero grade cytotoxicity as quantified by MTT assay. The fluorescent labeling process with our newly designed glycoprotein CPC is exceptionally efficient.

  16. Splenic scintigraphy using Tc-99m-labeled heat-denatured red blood cells in pediatric patients: concise communication

    SciTech Connect

    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.

  17. Label-free and noninvasive optical detection of the distribution of nanometer-size mitochondria in single cells

    NASA Astrophysics Data System (ADS)

    Su, Xuantao; Qiu, Yuanyuan; Marquez-Curtis, Leah; Gupta, Manisha; Capjack, Clarence E.; Rozmus, Wojciech; Janowska-Wieczorek, Anna; Tsui, Ying Y.

    2011-06-01

    A microfluidic flow cytometric technique capable of obtaining information on nanometer-sized organelles in single cells in a label-free, noninvasive optical manner was developed. Experimental two-dimensional (2D) light scattering patterns from malignant lymphoid cells (Jurkat cell line) and normal hematopoietic stem cells (cord blood CD34+ cells) were compared with those obtained from finite-difference time-domain simulations. In the simulations, we assumed that the mitochondria were randomly distributed throughout a Jurkat cell, and aggregated in a CD34+ cell. Comparison of the experimental and simulated light scattering patterns led us to conclude that distinction from these two types of cells may be due to different mitochondrial distributions. This observation was confirmed by conventional confocal fluorescence microscopy. A method for potential cell discrimination was developed based on analysis of the 2D light scattering patterns. Potential clinical applications using mitochondria as intrinsic biological markers in single cells were discussed in terms of normal cells (CD34+ cell and lymphocytes) versus malignant cells (THP-1 and Jurkat cell lines).

  18. FM dyes label sterol-rich plasma membrane domains and are internalized independently of the cytoskeleton in characean internodal cells.

    PubMed

    Klima, Andreas; Foissner, Ilse

    2008-10-01

    We applied the endocytic markers FM1-43, FM4-64 and filipin to internodal cells of the green alga Chara corallina. Both FM dyes stained stable, long-living plasma membrane patches with a diameter of up to 1 microm. After 5 min, FM dyes labeled cortical, trembling structures up to 500 nm in size. After 15 min, FM dyes localized to endoplasmic organelles up to 1 microm in diameter, which migrated actively along actin bundles or participated in cytoplasmic mass streaming. After 30-60 min, FM fluorescence appeared in the membrane of small, endoplasmic vacuoles but not in that of the central vacuole. Some of the FM-labeled organelles were also stained by neutral red and lysotracker yellow, indicative of acidic compartments. Filipin, a sterol-specific marker, likewise labeled plasma membrane domains which co-localized with the FM patches. However, internalization of filipin could not be observed. KCN, cytochalasin D, latrunculin B and oryzalin had no effect on size, shape and distribution of FM- and filipin-labeled plasma membrane domains. Internalization of FM dyes was inhibited by KCN but not by drugs which interfere with the actin or microtubule cytoskeleton. Our data indicate that the plasma membrane of characean internodal cells contains discrete domains which are enriched in sterols and probably correspond to clusters of lipid rafts. The inhibitor experiments suggest that FM uptake is active but independent of actin filaments, actin polymerization and microtubules. The possible function of the sterol-rich, FM labeled plasma membrane areas and the significance of actin-independent FM internalization (via endocytosis or energy-dependent flippases) are discussed.

  19. Superparamagnetic Iron Oxide Nanoparticles as MRI contrast agents for Non-invasive Stem Cell Labeling and Tracking

    PubMed Central

    Li, Li; Jiang, Wen; Luo, Kui; Song, Hongmei; Lan, Fang; Wu, Yao; Gu, Zhongwei

    2013-01-01

    Stem cells hold great promise for the treatment of multiple human diseases and disorders. Tracking and monitoring of stem cells in vivo after transplantation can supply important information for determining the efficacy of stem cell therapy. Magnetic resonance imaging (MRI) combined with contrast agents is believed to be the most effective and safest non-invasive technique for stem cell tracking in living bodies. Commercial superparamagnetic iron oxide nanoparticles (SPIONs) in the aid of transfection agents (TAs) have been applied to labeling stem cells. However, owing to the potential toxicity of TAs, more attentions have been paid to develop novel SPIONs with specific surface coating or functional moieties which facilitate effective cell internalization in the absence of TAs. This review aims to summarize the recent progress in the design and preparation of SPIONs as cellular MRI probes, to discuss their applications and current problems facing in stem cell labeling and tracking, and to offer perspectives and solutions for the future development of SPIONs in this field. PMID:23946825

  20. Label-free electrical discrimination of cells at normal, apoptotic and necrotic status with a microfluidic device.

    PubMed

    Gou, Hong-Lei; Zhang, Xian-Bo; Bao, Ning; Xu, Jing-Juan; Xia, Xing-Hua; Chen, Hong-Yuan

    2011-08-19

    As a label-free alternative of conventional flow cytometry, chip-based impedance measurement for single cell analysis has attracted increasing attentions in recent years. In this paper, we designed a T-shape microchannel and fabricated a pair of gold electrodes located horizontally on each side of the microchannel using a transfer printing method. Instant electric signals of flowing-through single cells were then detected by connecting the electrodes to a Keithley resistance and capacitance measurement system. Experimental results based on the simultaneous measurement of resistance and capacitance demonstrated that HL-60 and SMMC-7721 cells could be differentiated effectively. Moreover, SMMC-7721 cells at normal, apoptotic and necrotic status can also be discriminated in the flow. We discussed the possible mechanism for the discrimination of cell size and cell status by electrical analysis, and it is believed that the improvement of detection with our design results from more uniform distribution of the electric field. This microfluidic design may potentially become a promising approach for the label-free cell sorting and screening.

  1. Therapeutics with SPION-labeled stem cells for the main diseases related to brain aging: a systematic review

    PubMed Central

    Alvarim, Larissa T; Nucci, Leopoldo P; Mamani, Javier B; Marti, Luciana C; Aguiar, Marina F; Silva, Helio R; Silva, Gisele S; Nucci-da-Silva, Mariana P; DelBel, Elaine A; Gamarra, Lionel F

    2014-01-01

    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. PMID:25143726

  2. [Effects of superparamagnetic iron-oxide particles-labeling on the multi-diffentiation of rabbit marrow mesenchymal stem cell in vitro].

    PubMed

    Jin, Xuhong; Yang, Liu; Zhang, Shou; Dun, Xiaojun; Wang, Fuyou; Tan, Hongbo

    2012-02-01

    The aim of this study was to label rabbit bone derived mesenchymal stem cells (BMSCs) with superparamagnetic iron oxide particles (SPIO) and to study the effects of magnetic labeling on the multi-differentiation of BMSCs. Rabbit BMSCs were isolated, purified, expanded, then coincubated with SPIO(25 microg/ml) complexed to protamine sulfate (Pro) transfection agents overnight. Prussian blue staining and transmission electron microscopy were performed to show intracellular iron. Cell differentiation was evaluated. Both labeled and unlabeled BMSCs were subjected to osteogenic, adipogenic and chondrogenic differentiation to assess their differentiation capacity for 21 d. Osteogenic cells were stained with alizarin red to reveal calcium deposition, adipogenic cells were stained with oil redO' respectively. Chondrogenic cells stained with Safranin-O, glycosamino glycans, and type II collagen production was assessed by standard immunohistochemistry. Cell with immunohistochemistry staining were detected by polarized light microscopy and analysed by Image-Pro Plus software. The results showed that intracytoplasmic nanoparticles were stained with Prussian blue and observed by transmission electron microscopy clearly except the unlabeled control. As compared with the nonlabeled cells, it showed no statistically significant difference on the differentiation of the labeled BMSCs. And the differentiation of the labeled cells were unaffected by the endosomal incorporation of SPIO. In summary, BMSCs can be labeled with SPIO without significant change in cell multi-differentiation capacity. PMID:22404022

  3. Quantitative analysis of cell surface membrane proteins using membrane-impermeable chemical probe coupled with 18O labeling

    PubMed Central

    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

  4. Neuron-specific enolase-like immunoreactivity in the vertebrate retina: selective labelling of Müller cells in Anura.

    PubMed

    Wilhelm, M; Straznicky, C; Gábriel, R

    1992-11-01

    Neuron-specific enolase (NSE) immunocytochemistry was carried out in retinae of goldfish, axolotl, clawed frog, cane toad, lizard, chick, guinea-pig, rabbit, rat, cat and human. With the exception of Anura, strong immunoreactivity was seen in the large ganglion, amacrine cells and horizontal cells of the retina in all of the other species. Photoreceptors were found to be labelled in the rat and human retina and only one cone type in rabbit. Photoreceptor pedicles and ellipsoids were stained in the goldfish and the somata and inner segments of some photoreceptors in axolotl. In the axolotl retina, besides neurons, Müller cells (MCs) were also immunolabelled. In the retina of the cane toad and the clawed frog MCs were the only stained elements. Similarly in other parts of the central nervous system of the cane toad, glial elements of the optic tectum and spinal cord were immunoreactive. In contrast, in the peripheral nervous system, neurons of the 1st sympathetic ganglion and the 2nd dorsal root ganglion were labelled. In double-labelling experiments, glial fibrillary acidic protein and NSE showed colocalisation both in the glial elements of the optic tectum and spinal cord and in MCs of the retina of the cane toad.

  5. The relationship between cell proliferation and differentiation and mapping of putative dental pulp stem/progenitor cells during mouse molar development by chasing BrdU-labeling.

    PubMed

    Ishikawa, Yuko; Ida-Yonemochi, Hiroko; Nakakura-Ohshima, Kuniko; Ohshima, Hayato

    2012-04-01

    Human dental pulp contains adult stem cells. Our recent study demonstrated the localization of putative dental pulp stem/progenitor cells in the rat developing molar by chasing 5-bromo-2'-deoxyuridine (BrdU)-labeling. However, there are no available data on the localization of putative dental pulp stem/progenitor cells in the mouse molar. This study focuses on the mapping of putative dental pulp stem/progenitor cells in addition to the relationship between cell proliferation and differentiation in the developing molar using BrdU-labeling. Numerous proliferating cells appeared in the tooth germ and the most active cell proliferation in the mesenchymal cells occurred in the prenatal stages, especially on embryonic Day 15 (E15). Cell proliferation in the pulp tissue dramatically decreased in number by postnatal Day 3 (P3) when nestin-positive odontoblasts were arranged in the cusped areas and disappeared after postnatal Week 1 (P1W). Root dental papilla included numerous proliferating cells during P5 to P2W. Three to four intraperitoneal injections of BrdU were given to pregnant ICR mice and revealed slow-cycling long-term label-retaining cells (LRCs) in the mature tissues of postnatal animals. Numerous dense LRCs postnatally decreased in number and reached a plateau after P1W when they mainly resided in the center of the dental pulp, associating with blood vessels. Furthermore, numerous dense LRCs co-expressed mesenchymal stem cell markers such as STRO-1 and CD146. Thus, dense LRCs in mature pulp tissues were believed to be dental pulp stem/progenitor cells harboring in the perivascular niche surrounding the endothelium.

  6. Comparison of label-free and GFP multiphoton imaging of hair follicle-associated pluripotent (HAP) stem cells in mouse whiskers.

    PubMed

    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.

  7. Infected and apoptotic cells in the IBDV-infected bursa of Fabricius, studied by double-labelling techniques.

    PubMed

    Nieper, H; Teifke, J P; Jungmann, A; Lohr, C V; Muller, H

    1999-06-01

    Infections of young chickens with infectious bursal disease virus (IBDV) result in depletion of lymphoid cells of the bursa of Fabricius (BF) due to necrosis and apoptotic processes. Interactions between IBDV and lymphoid cells were investigated by labelling paraffin-embedded tissue sections of infected BF with combinations of either immunohistochemistry (IHC), in situ hybridization (ISH) or in situ TUNEL reaction (IST). With regard to specificity and sensitivity, results of ISH were comparable to those of IHC. By double-labelling it was shown, for the first time, that viral antigen was present in most of the apoptotic cells. This suggests that IBDV may be directly involved in the induction of the apoptotic process. However, some cells also showed either viral antigen or DNA fragmentation, especially at the early stages of infection. It should be taken into account, therefore, that the apoptotic processes might also be induced by IBDV through indirect interaction between cells. Remarkably, in some of the infected lymphoid cells ISH signals were observed in the nucleolus. PMID:26915384

  8. Connective Tissue Growth Factor reporter mice label a subpopulation of mesenchymal progenitor cells that reside in the trabecular bone region.

    PubMed

    Wang, Wen; Strecker, Sara; Liu, Yaling; Wang, Liping; Assanah, Fayekah; Smith, Spenser; Maye, Peter

    2015-02-01

    Few gene markers selectively identify mesenchymal progenitor cells inside the bone marrow. We have investigated a cell population located in the mouse bone marrow labeled by Connective Tissue Growth Factor reporter expression (CTGF-EGFP). Bone marrow flushed from CTGF reporter mice yielded an EGFP+ stromal cell population. Interestingly, the percentage of stromal cells retaining CTGF reporter expression decreased with age in vivo and was half the frequency in females compared to males. In culture, CTGF reporter expression and endogenous CTGF expression marked the same cell types as those labeled using Twist2-Cre and Osterix-Cre fate mapping approaches, which previously had been shown to identify mesenchymal progenitors in vitro. Consistent with this past work, sorted CTGF+ cells displayed an ability to differentiate into osteoblasts, chondrocytes, and adipocytes in vitro and into