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Sample records for fepro cell labeling

  1. MRI Tracking of FePro Labeled Fresh and Cryopreserved Long Term In Vitro Expanded Human Cord Blood AC133+ Endothelial Progenitor Cells in Rat Glioma

    PubMed Central

    Janic, Branislava; Jafari-Khouzani, Kourosh; Babajani-Feremi, Abbas; Iskander, A. S. M.; Varma, Nadimpalli Ravi S.; Ali, Meser M.; Knight, Robert A.; Arbab, Ali S.

    2012-01-01

    Background Endothelial progenitors cells (EPCs) are important for the development of cell therapies for various diseases. However, the major obstacles in developing such therapies are low quantities of EPCs that can be generated from the patient and the lack of adequate non-invasive imaging approach for in vivo monitoring of transplanted cells. The objective of this project was to determine the ability of cord blood (CB) AC133+ EPCs to differentiate, in vitro and in vivo, toward mature endothelial cells (ECs) after long term in vitro expansion and cryopreservation and to use magnetic resonance imaging (MRI) to assess the in vivo migratory potential of ex vivo expanded and cryopreserved CB AC133+ EPCs in an orthotopic glioma rat model. Materials, Methods and Results The primary CB AC133+ EPC culture contained mainly EPCs and long term in vitro conditions facilitated the maintenance of these cells in a state of commitment toward endothelial lineage. At days 15–20 and 25–30 of the primary culture, the cells were labeled with FePro and cryopreserved for a few weeks. Cryopreserved cells were thawed and in vitro differentiated or IV administered to glioma bearing rats. Different groups of rats also received long-term cultured, magnetically labeled fresh EPCs and both groups of animals underwent MRI 7 days after IV administration of EPCs. Fluorescent microscopy showed that in vitro differentiation of EPCs was not affected by FePro labeling and cryopreservation. MRI analysis demonstrated that in vivo accumulation of previously cryopreserved transplanted cells resulted in significantly higher R2 and R2* values indicating a higher rate of migration and incorporation into tumor neovascularization of previously cryopreserved CB AC133+ EPCs to glioma sites, compared to non-cryopreserved cells. Conclusion Magnetically labeled CB EPCs can be in vitro expanded and cryopreserved for future use as MRI probes for monitoring the migration and incorporation to the sites of

  2. 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

  3. Monitoring ferumoxide-labelled neural progenitor cells and lesion evolution by magnetic resonance imaging in a model of cell transplantation in cerebral ischaemia

    PubMed Central

    2014-01-01

    Efficacy of neural stem/progenitor cell (NPC) therapies after cerebral ischaemia could be better evaluated by monitoring in vivo migration and distribution of cells post-engraftment in parallel with analysis of lesion volume and functional recovery. Magnetic resonance imaging (MRI) is ideally placed to achieve this, but still poses several challenges. We show that combining the ferumoxide MRI contrast agent Endorem with protamine sulphate (FePro) improves iron oxide uptake in cells compared to Endorem alone and is non-toxic. Hence FePro complex is a better contrast agent than Endorem for monitoring NPCs. FePro complex-labelled NPCs proliferated and differentiated normally in vitro, and upon grafting into the brain 48 hours post-ischaemia they were detected in vivo by MRI. Imaging over four weeks showed the development of a confounding endogenous hypointense contrast evolution at later timepoints within the lesioned tissue. This was at least partly due to accumulation within the lesion of macrophages and endogenous iron. Neither significant NPC migration, assessed by MRI and histologically, nor a reduction in the ischaemic lesion volume was observed in NPC-grafted brains.  Crucially, while MRI provides reliable information on engrafted cell location early after an ischaemic insult, pathophysiological changes to ischaemic lesions can interfere with cellular imaging at later timepoints. PMID:24715962

  4. 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. PMID:18465447

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. Isotope Labeling in Mammalian Cells

    PubMed Central

    Dutta, Arpana; Saxena, Krishna; Klein-Seetharaman, Judith

    2011-01-01

    Isotope labeling of proteins represents an important and often required tool for the application of nuclear magnetic resonance (NMR) spectroscopy to investigate the structure and dynamics of proteins. Mammalian expression systems have conventionally been considered to be too weak and inefficient for protein expression. However, recent advances have significantly improved the expression levels of these systems. Here, we provide an overview of some of the recent developments in expression strategies for mammalian expression systems in view of NMR investigations. PMID:22167668

  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. Cells labeled with multiple fluorophores bound to a nucleic acid carrier

    SciTech Connect

    Dattagupta, N.; Kamarch, M.E.

    1989-04-25

    In passing labeled cells through a cell sorter, the improvement which comprises employing a labeled cell comprising a cell, an antibody specific to and bound to such cell, a nucleic acid fragment joined to the antibody, and a plurality of labels on the nucleic acid fragment. Because of the presence of multiple labels, the sensitivity of the separation of labeled cells in increased.

  12. 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.

  13. High efficiency labeling of glycoproteins on living cells

    PubMed Central

    Zeng, Ying; Ramya, T. N. C.; Dirksen, Anouk; Dawson, Philip E.; Paulson, James C.

    2010-01-01

    We describe a simple method for efficiently labeling cell surface glycans on virtually any living animal cell. The method employs mild Periodate oxidation to generate an aldehyde on sialic acids, followed by Aniline-catalyzed oxime Ligation with a suitable tag (PAL). Aniline catalysis dramatically accelerates oxime ligation, allowing use of low concentrations of aminooxy-biotin at neutral pH to label the majority of cell surface glycoproteins while maintaining high cell viability. PMID:19234450

  14. 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.

  15. 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

  16. Identification and behavior of label-retaining cells in epithelia

    SciTech Connect

    Bickenbach, J.R.

    1982-01-01

    A subpopulation of stem cells has been demonstrated in several renewing tissues. Such cells have a slow cell cycle and provide differentiating cells during normal turnover and during regeneration of the tissue following damage. The presence of slowly-cycling cells in epithelia from regions of skin and oral mucosa was examined by labeling 10-day-old mice and 5-day-old hamsters with tritiated thymidine (/sup 3/H-TdR) and observing the rate at which label was diluted from the basal cells. Label was rapidly diluted by cell division in most cells but a small percentage of basal cells (label-retaining cells, LRCS) was found to retain label for up to ninety days. Electron microscopic autoradiography and ..beta..-glucuronidase histochemistry with autoradiography were used to distinguish slowly-cycling keratinocytes from Langerhans cells. Such findings of slowly-cycling keratinocytes in epithelia with the ability to proliferate in culture and with a direct relationship to patterns of tissue architecture suggest that LRCs in epithelia correspond to stem cells described in other continuously renewing tissues.

  17. Label-free high-throughput cell screening in flow

    PubMed Central

    Mahjoubfar, Ata; Chen, Claire; Niazi, Kayvan R.; Rabizadeh, Shahrooz; Jalali, Bahram

    2013-01-01

    Flow cytometry is a powerful tool for cell counting and biomarker detection in biotechnology and medicine especially with regards to blood analysis. Standard flow cytometers perform cell type classification both by estimating size and granularity of cells using forward- and side-scattered light signals and through the collection of emission spectra of fluorescently-labeled cells. However, cell surface labeling as a means of marking cells is often undesirable as many reagents negatively impact cellular viability or provide activating/inhibitory signals, which can alter the behavior of the desired cellular subtypes for downstream applications or analysis. To eliminate the need for labeling, we introduce a label-free imaging-based flow cytometer that measures size and cell protein concentration simultaneously either as a stand-alone instrument or as an add-on to conventional flow cytometers. Cell protein concentration adds a parameter to cell classification, which improves the specificity and sensitivity of flow cytometers without the requirement of cell labeling. This system uses coherent dispersive Fourier transform to perform phase imaging at flow speeds as high as a few meters per second. PMID:24049682

  18. Live cell immunogold labelling of RNA polymerase II

    PubMed Central

    Orlov, Igor; Schertel, Andreas; Zuber, Guy; Klaholz, Bruno; Drillien, Robert; Weiss, Etienne; Schultz, Patrick; Spehner, Danièle

    2015-01-01

    Labeling nuclear proteins with electron dense probes in living cells has been a major challenge due to their inability to penetrate into nuclei. We developed a lipid-based approach for delivering antibodies coupled to 0.8 nm ultrasmall gold particles into the nucleus to label RNA polymerase II. Focussed Ion Beam slicing coupled to Scanning Electron Microscopy (FIB/SEM) enabled visualization of entire cells with probe localization accuracy in the 10 nm range. PMID:25662860

  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. PMID:1030938

  20. 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.

  1. Use of indium-111 as a red cell label

    SciTech Connect

    AuBuchon, J.P.; Brightman, A.

    1989-02-01

    To select the most promising 111In chelate for use as a second red cell (RBC) label for comparison of the survival of autologous and allogeneic cells, 49 normal RBC samples were studied in vitro after being labeled with 111In-8-hydroxyquinolinol (111In-oxine) prepared by three different methods, 111In-tropolone, and 111In-acetylacetone. Labeling efficiencies reached 99 percent and did not decline when the amount of 111In used was increased from 1.75 to 50 muCi per ml of RBCs. Storage of labeled RBCs in normal AB plasma at 4, 22, and 37 degrees C for up to 48 hours resulted in a similar rate of loss of the label from the RBCs with all labeling methods. These rates were time- and temperature-dependent and were accurate predictions of the rates found in later in vivo experimentation. Fresh RBCs from 11 subjects were labeled with 111In chelated with oxine in the presence of the RBCs or chelated with tropolone just prior to the labeling. RBC mass determinations using these autologous RBCs labeled with 111In accurately reflected the subjects' RBC masses as predicted through standard morphometric formulae. The rate of disappearance of the radionuclide after reinfusion of the autologous RBCs decreased with time. At 24 hours after reinfusion, 89.5 +/- 1.29 percent (mean +/- SEM) of the 111In-tropolone and 87.3 +/- 1.25 percent of the 111In-oxine continued in circulation. 111In is a simple and efficient agent for the labeling of RBCs for blood volume determinations and short-term survivals.

  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. Fluorescent Labeling of Yeast Cell Wall Components.

    PubMed

    Okada, Hiroki; Ohya, Yoshikazu

    2016-01-01

    Yeast cells stained with a fluorescent dye that specifically binds to one of the cell wall components can be observed under a fluorescent microscope. Visualization of the components 1,3-β-glucan, mannoproteins, and/or chitin not only provides information concerning the cell wall, but also reveals clues about various cellular activities such as cell polarity, vesicular transport, establishment of budding pattern, apical and isotropic bud growth, and replicative cell age. This protocol describes a standard method for visualizing different components of the yeast cell wall. PMID:27480714

  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. 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. PMID:25536421

  6. Interfacial Polymerization for Colorimetric Labeling of Protein Expression in Cells

    PubMed Central

    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. PMID:25536421

  7. Labeling proteins on live mammalian cells using click chemistry.

    PubMed

    Nikić, Ivana; Kang, Jun Hee; Girona, Gemma Estrada; Aramburu, Iker Valle; Lemke, Edward A

    2015-05-01

    We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced--by site-directed mutagenesis--at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4-7 d, followed by the described transfections and labeling reaction steps, which can take 3-4 d. PMID:25906116

  8. Imaging of inflammatory processes with labeled cells

    SciTech Connect

    Froelich, J.W.; Swanson, D.

    1984-04-01

    Radionuclide techniques for localizing inflammatory processes had relied heavily upon /sup 67/Ga-citrate until McAfee and Thakur described the technique for the radiolabeling of leukocytes with /sup 111/In-oxine. Since their initial description in 1976 there has been continued development of the radiopharmaceutical, as well as clinical efficacy. At present /sup 111/In-labeled leukocytes continue to be handled as an investigational new drug but this has not greatly limited its clinical availability. Indium-/sup 111/ leukocytes are the agent of choice for evaluation of patients with fever of unknown origin, osteomyelitis, and prosthetic graft infections; and preliminary data shows great promise in the area of detecting reoccurrence of inflammatory bowel disease. This article attempts to review currently accepted uses of 111In leukocytes as well as potential areas of application.

  9. Water soluble fluorescence quantum dot probe labeling liver cancer cells.

    PubMed

    Chang, Baoxing; Yang, Xianjun; Wang, Fang; Wang, Yinsong; Yang, Rui; Zhang, Ning; Wang, Baiqi

    2013-11-01

    Water soluble quantum dots (QDs) have been prepared by hydrothermal method and characterized by ultraviolet irradiation, XRD, TEM, UV-Vis absorption spectrometer and fluorescence spectrometer. Then the QD-antibody-AFP probes (QD-Ab-AFP) were synthesized by chemical process and specifically labeled AFP antigen in PLC/PRF/5 liver cancer cells. The results showed that the QDs were cubic structure and had excellent optical properties. Moreover, the QD-Ab-AFP with good stability could specifically label liver cancer cells. This work provides strong foundation for further studying and developing new approach to detect liver cancer at early stage. PMID:23888351

  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

    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

  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

    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.

  14. Separating Magnetically Labeled and Unlabeled Biological Cells within Microfluidic Channels

    NASA Astrophysics Data System (ADS)

    Byvank, Tom; Vieira, Greg; Miller, Brandon; Yu, Bo; Chalmers, Jeffrey; Lee, L. James; Sooryakumar, R.

    2011-03-01

    The transport of microscopic objects that rely on magnetic forces have numerous advantages including flexibility of controlling many design parameters and the long range magnetic interactions generally do not adversely affect biological or chemical interactions. We present results on the use of magnetic micro-arrays imprinted within polydimethylsiloxane (PDMS) microfluidic channels that benefit from these features and the ability to rapidly reprogram the magnetic energy landscape for cell manipulation and sorting applications. A central enabling feature is the very large, tunable, magnetic field gradients (> 10 4) that can be designed within the microfluidic architecture. Through use of antibody-conjugated magnetic microspheres to label biological cells, results on the transport and sorting of heterogeneous cell populations are presented. The effects of micro-array and fluid channel design parameters, competition between magnetic forces and hydrodynamic drag forces, and cell-labeling efficiency on cell separation are discussed.

  15. Multiplex cell and lineage tracking with combinatorial labels.

    PubMed

    Loulier, Karine; Barry, Raphaëlle; Mahou, Pierre; Le Franc, Yann; Supatto, Willy; Matho, Katherine S; Ieng, Siohoi; Fouquet, Stéphane; Dupin, Elisabeth; Benosman, Ryad; Chédotal, Alain; Beaurepaire, Emmanuel; Morin, Xavier; Livet, Jean

    2014-02-01

    We present a method to label and trace the lineage of multiple neural progenitors simultaneously in vertebrate animals via multiaddressable genome-integrative color (MAGIC) markers. We achieve permanent expression of combinatorial labels from new Brainbow transgenes introduced in embryonic neural progenitors with electroporation of transposon vectors. In the mouse forebrain and chicken spinal cord, this approach allows us to track neural progenitor's descent during pre- and postnatal neurogenesis or perinatal gliogenesis in long-term experiments. Color labels delineate cytoarchitecture, resolve spatially intermixed clones, and specify the lineage of astroglial subtypes and adult neural stem cells. Combining colors and subcellular locations provides an expanded marker palette to individualize clones. We show that this approach is also applicable to modulate specific signaling pathways in a mosaic manner while color-coding the status of individual cells regarding induced molecular perturbations. This method opens new avenues for clonal and functional analysis in varied experimental models and contexts. PMID:24507188

  16. Specific cell surface labeling of GPCRs using split GFP.

    PubMed

    Jiang, Wen-Xue; Dong, Xu; Jiang, Jing; Yang, Yu-Hong; Yang, Ju; Lu, Yun-Bi; Fang, San-Hua; Wei, Er-Qing; Tang, Chun; Zhang, Wei-Ping

    2016-01-01

    Specific cell surface labeling is essential for visualizing the internalization processes of G-protein coupled receptors (GPCRs) and for gaining mechanistic insight of GPCR functions. Here we present a rapid, specific, and versatile labeling scheme for GPCRs at living-cell membrane with the use of a split green fluorescent protein (GFP). Demonstrated with two GPCRs, GPR17 and CysLT2R, we show that two β-stands (β-stands 10 and 11) derived from a superfolder GFP (sfGFP) can be engineered to one of the three extracellular loop of a GPCR. The complementary fragment of sfGFP has nine β-strands (β-stands 1-9) that carries the mature fluorophore, and can be proteolytically derived from the full-length sfGFP. Separately the GFP fragments are non-fluorescent, but become fluorescent upon assembly, thus allowing specific labeling of the target proteins. The two GFP fragments rapidly assemble and the resulting complex is extremely tight under non-denaturing conditions, which allows real-time and quantitative assessment of the internalized GPCRs. We envision that this labeling scheme will be of great use for labeling other membrane proteins in various biological and pharmacological applications. PMID:26857153

  17. 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.

  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. Labeling Stem Cells with Superparamagnetic Iron Oxide Nanoparticles: Analysis of the Labeling Efficacy by Microscopy and Magnetic Resonance Imaging

    PubMed Central

    Jasmin; Torres, Ana Luiza Machado; Jelicks, Linda; de Carvalho, Antonio Carlos Campos; Spray, David C.; Mendez-Otero, Rosalia

    2013-01-01

    Stem cell therapy has emerged as a potential therapeutic option for cell death-related heart diseases. Application of non-invasive cell tracking approaches is necessary to determine tissue distribution and lifetime of stem cells following their injection and will likely provide knowledge about poorly understood stem cells mechanisms of tissue repair. Magnetic resonance imaging (MRI) is a potentially excellent tool for high-resolution visualization of the fate of cells after transplantation and for evaluation of therapeutic strategies. The application of MRI for in vivo cell tracking requires contrast agents to achieve efficient cell labeling without causing any toxic cellular effects or eliciting any other side effects. For these reasons clinically approved contrast agents (e.g., ferumoxides) and incorporation facilitators (e.g., protamine) are currently the preferred materials for cell labeling and tracking. Here we describe how to use superparamag-netic iron oxide nanoparticles to label cells and to monitor cell fate in several disease models. PMID:22791437

  20. Investigations into agents for improving cell labeling with positron- and gamma-emitting radionuclides

    SciTech Connect

    Zoghbi, S.S.; Thakur, M.L.; Gottschalk, A.; Pande, S.; Srivastava, S.C.; Richards, P.

    1982-01-01

    It was possible to label leukocytes with Co-oxine, but a large proportion of the radioactivity was eluted from the cells upon washing. Ruthenium oxine labeled platelets efficiently in plasma while negligible proportion of radioactivity was eluted from the cells. Three factors influence the labeling efficiency of the cells: duration of the incubation periods; cell concentration; and ACD concentration.

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

    NASA Astrophysics Data System (ADS)

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

    2010-05-01

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

  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. Fluorescent labeling of tetracysteine-tagged proteins in intact cells

    PubMed Central

    Hoffmann, Carsten; Gaietta, Guido; Zürn, Alexander; Adams, Stephen R; Terrillon, Sonia; Ellisman, Mark H; Tsien, Roger Y; Lohse, Martin J

    2011-01-01

    In this paper, we provide a general protocol for labeling proteins with the membrane-permeant fluorogenic biarsenical dye fluorescein arsenical hairpin binder–ethanedithiol (FlAsH-EDT2). Generation of the tetracysteine-tagged protein construct by itself is not described, as this is a protein-specific process. This method allows site-selective labeling of proteins in living cells and has been applied to a wide variety of proteins and biological problems. We provide here a generally applicable labeling procedure and discuss the problems that can occur as well as general considerations that must be taken into account when designing and implementing the procedure. The method can even be applied to proteins with expression below 1 pmol mg−1 of protein, such as G protein–coupled receptors, and it can be used to study the intracellular localization of proteins as well as functional interactions in fluorescence resonance energy transfer experiments. The labeling procedure using FlAsH-EDT2 as described takes 2–3 h, depending on the number of samples to be processed. PMID:20885379

  5. 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

  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. A self-assembling fluorescent dipeptide conjugate for cell labelling.

    PubMed

    Kirkham, Steven; Hamley, Ian W; Smith, Andrew M; Gouveia, Ricardo M; Connon, Che J; Reza, Mehedi; Ruokolainen, Janne

    2016-01-01

    Derivatives of fluorophore FITC (fluorescein isothiocyanate) are widely used in bioassays to label proteins and cells. An N-terminal leucine dipeptide is attached to FITC, and we show that this simple conjugate molecule is cytocompatible and is uptaken by cells (human dermal and corneal fibroblasts) in contrast to FITC itself. Co-localisation shows that FITC-LL segregates in peri-nuclear and intracellular vesicle regions. Above a critical aggregation concentration, the conjugate is shown to self-assemble into beta-sheet nanostructures comprising molecular bilayers. PMID:25990811

  9. 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

  10. 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.

  11. A new model to simulate and analyze proliferating cell populations in BrdU labeling experiments

    PubMed Central

    2013-01-01

    Background This paper presents a novel model for proliferating cell populations in labeling experiments. It is especially tailored to the technique of Bromodeoxyuridine (BrdU), which is taken up by dividing cells and thus accumulates with increasing division number during uplabeling. The study of the evolving label intensities of BrdU labeled cell populations is aimed at quantifying proliferation properties such as division and death rates. Results In contrast to existing models, our model considers a labeling efficacy that follows a distribution, rather than a uniform value. It thereby allows to account for noise as well as possibly space-dependent heterogeneity in the effective label uptake of the individual cells in a population. Furthermore, it enables more informative comparison with experimental data: The population-level label distribution is provided as a model output, thereby increasing the information content compared to existing models that give the fraction of labeled cells or the mean label intensity. We employ our model to study some naturally arising examples of heterogeneity in label uptake, which are not covered by existing models. With simulations of noisy and spacially heterogeneous label uptake, we demonstrate that our model contributes a more realistic quantitative description of labeling experiments. Conclusion The presented model is to our knowledge the first one that predicts the full label distribution for BrdU labeling experiments. Thus, it can exploit more information, namely the full intensity distribution, from labeling measurements, and thereby opens up new quantitative insights into cell proliferation. PMID:24268033

  12. 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.

  13. Enhanced detection of fluorescence quenching in labeled cells

    SciTech Connect

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

    1992-01-28

    This patent describes a method for relatively quantifying BrdU-labeled DNA in cells in an S-phase during a selected interval within a cell cycle. It comprises: incorporating the BrdU into the DNA for a time of about 5 minutes to thirty minutes; staining the DNA with a first fluorochrome having a fluorescence which is quenchable by BrdU; staining the DNA with a second fluorochrome having a fluorescence which is substantially unaffected by BrdU; measuring fluorescence from the first and second fluorochromes to obtain first and second output signals, respectively, on a cell-by-cell basis; and subtracting the first output signal from the second output signal to obtain a different signal functionally related to the quantity of incorporated BrdU.

  14. 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

  15. Label-free detection of immune complexes with myeloid cells.

    PubMed

    Szittner, Z; Bentlage, A E H; Rovero, P; Migliorini, P; Lóránd, V; Prechl, J; Vidarsson, G

    2016-07-01

    The aim of this study was to provide proof-of-concept for quantitative and qualitative label-free detection of immune complexes through myeloid cells with imaging surface plasmon resonance. Surface plasmon resonance imaging was first applied to monitor the binding of human sera from healthy and rheumatoid arthritis (RA) patients to immobilized citrullinated RA-specific peptide antigens, histone citrullinated peptide 2 (HCP2) and viral citrullinated peptide 2 (VCP2). Next, the binding of monocytoid cell line U937 to the resulting immune complexes on the sensor surface was monitored. As control, binding of U937 was monitored to immunoglobulin (Ig)G subclasses simultaneously. Cell response results were compared to results of cyclic citrullinated peptide 2 (CCP2) enzyme-linked immunosorbent assay (ELISA), clinical RA diagnosis and antigen-specific antibody distribution of the samples. Human IgG3 triggered the most pronounced response, followed by IgG1 and IgG4, while IgG2 did not result in U937 cell binding. Serum samples obtained from RA patients resulted in a significantly increased cell response to VCP2 compared to healthy controls. The strength of cell response towards VCP2 immune complexes showed significant correlation with levels of antigen-specific IgA, IgG and IgG3. Cellular responses on VCP2 immune complexes showed significant association with both CCP2-based serological positivity and European League Against Rheumatism (EULAR) criteria-based clinical RA diagnosis. Immunoglobulin-triggered binding of monocytoid cells can be monitored using a label-free multiplex technology. Because these binding events are presumably initiated by Fc receptors, the system provides a tool for biological detection of autoantibodies with diagnostic value, here exemplified by anti-citrullinated antibodies. This provides added information to antibody levels, as interaction with Fc-receptor-expressing cells is also affected by post-translational modification of the immunoglobulins

  16. 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

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

    PubMed Central

    Rhim, Andrew D.

    2015-01-01

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

  18. Evaluating the efficacy of subcellular fractionation of blast cells using live cell labeling and 2D DIGE.

    PubMed

    Ho, Yin Ying; Penno, Megan; Perugini, Michelle; Lewis, Ian; Hoffmann, Peter

    2012-01-01

    Labeling of exposed cell surface proteins of live cells using CyDye DIGE fluor minimal dyes is an efficient strategy for cell surface proteome profiling and quantifying differentially expressed proteins in diseases. Here we describe a strategy to evaluate a two-step detergent-based protein fractionation method using live cell labeling followed by visualization of the fluorescently labeled cell surface proteins and fractionated proteins within a single 2D gel. PMID:22311770

  19. 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).

  20. Single-cell label-free photoacoustic flowoxigraphy in vivo

    PubMed Central

    Wang, Lidai; Maslov, Konstantin; Wang, Lihong V.

    2013-01-01

    Label-free functional imaging of single red blood cells (RBCs) in vivo holds the key to uncovering the fundamental mechanism of oxygen metabolism in cells. To this end, we developed single-RBC photoacoustic flowoxigraphy (FOG), which can image oxygen delivery from single flowing RBCs in vivo with millisecond-scale temporal resolution and micrometer-scale spatial resolution. Using intrinsic optical absorption contrast from oxyhemoglobin (HbO2) and deoxyhemoglobin (HbR), FOG allows label-free imaging. Multiple single-RBC functional parameters, including total hemoglobin concentration (CHb), oxygen saturation (sO2), sO2 gradient (), flow speed (vf), and oxygen release rate (rO2), have been quantified simultaneously in real time. Working in reflection instead of transmission mode, the system allows minimally invasive imaging at more anatomical sites. We showed the capability to measure relationships among sO2, , vf, and rO2 in a living mouse brain. We also demonstrated that single-RBC oxygen delivery was modulated by changing either the inhalation gas or blood glucose. Furthermore, we showed that the coupling between neural activity and oxygen delivery could be imaged at the single-RBC level in the brain. The single-RBC functional imaging capability of FOG enables numerous biomedical studies and clinical applications. PMID:23536296

  1. 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.

  2. 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

  3. 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

  4. 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.

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

    PubMed

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

    2016-09-01

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

  6. 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. PMID:26234504

  7. Flow cytometry-based characterization of label-retaining stem cells following transplacental BrdU labelling.

    PubMed

    Poojan, Shiv; Kumar, Sushil

    2011-02-01

    A method to characterize and culture stem cells from neonate mouse epidermis after transplacental BrdU (bromo-deoxyuridine) administration is described. We have characterized stem cells by their properties viz. to retain BrdU label, adhere rapidly onto collagen-fibronectin substratum and express a specific biomarker beta-1-integrin. BrdU-labelled cells (detected using monoclonal antibody) constituted a sum of 18% of the total number of cells. The ability of freshly isolated keratinocytes [LRCs (label-retaining cells)] to bind to primary BrdU antibody or to pick up PI (propidium iodide) stain was distinguishable. Viable LRCs did not retain PI. Such cells, termed EpSC (epidermis stem cell), were PI negative and BrdU positive. EpSC constituted 6% of the total cell yield. Culture in low Ca2+ medium and susceptibility to differentiation in the presence of high Ca2+ levels further characterized the stem cells. This protocol is useful for studying transplacental carcinogenesis. PMID:21261598

  8. Genetic Method for Labeling Electrically Coupled Cells: Application to Retina

    PubMed Central

    Qiao, Mu; Sanes, Joshua R.

    2016-01-01

    Understanding how the nervous system functions requires mapping synaptic connections between neurons. Several methods are available for imaging neurons connected by chemical synapses, but few enable marking neurons connected by electrical synapses. Here, we demonstrate that a peptide transporter, Pept2, can be used for this purpose. Pept2 transports a gap junction-permeable fluorophore-coupled dipeptide, beta-alanine-lysine-N-7-amino-4-methyl coumarin-3-acid (βALA). Cre-dependent expression of pept2 in specific neurons followed by incubation in βALA labeled electrically coupled synaptic partners. Using this method, we analyze light-dependent modulation of electrical connectivity among retinal horizontal cells. PMID:26778956

  9. Long-term stem cell labeling by collagen-functionalized single-walled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Mao, Hongli; Cai, Rong; Kawazoe, Naoki; Chen, Guoping

    2014-01-01

    The monitoring of grafted stem cells is crucial to assess the efficiency, effectiveness and safety of such stem cell-based therapies. In this regard, a reliable and cytocompatible labeling method for stem cells is critically needed. In this study, the collagen-functionalized single-walled carbon nanotubes (Col-SWCNTs) were used as imaging probes for labeling of human mesenchymal stem cells (hMSCs) and the inherent Raman scattering of SWCNTs was used to image the SWCNT-labeled cells. The results showed that the Col-SWCNTs exhibit efficient cellular internalization by hMSCs without affecting their proliferation and differentiation. The prolonged dwell time of Col-SWCNTs in cells ensured the long-term labeling for up to 2 weeks. This work reveals the potential of Col-SWCNTs as probes for long-term stem cell labeling.

  10. 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...

  11. 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.

  12. 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.

  13. Effect of different magnetic nanoparticle coatings on the efficiency of stem cell labeling

    NASA Astrophysics Data System (ADS)

    Horák, Daniel; Babič, Michal; Jendelová, Pavla; Herynek, Vít; Trchová, Miroslava; Likavčanová, Katarina; Kapcalová, Miroslava; Hájek, Milan; Syková, Eva

    2009-05-01

    Maghemite nanoparticles with various coatings were prepared by the coprecipitation method and characterized by transmission electron microscopy, dynamic light scattering and IR in terms of morphology, size, polydispersity and surface coating. The labeling efficiency and the viability of both rat and human mesenchymal stem cells labeled with Endorem ®, poly( L-lysine) (PLL)-modified Endorem ®, uncoated γ-Fe 2O 3, D-mannose-, PLL- or poly( N,N-dimethylacrylamide) (PDMAAm)-coated γ-Fe 2O 3 nanoparticles were compared. Coated γ-Fe 2O 3 nanoparticles labeled cells better than did Endorem ®. High relaxation rates and in vitro magnetic resonance imaging of cells labeled with coated nanoparticles showed clearly visible contrast compared with unlabeled cells or cells labeled with Endorem ®.

  14. 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.

  15. Snap-, CLIP- and Halo-Tag Labelling of Budding Yeast Cells

    PubMed Central

    Stagge, Franziska; Mitronova, Gyuzel Y.; Belov, Vladimir N.; Wurm, Christian A.; Jakobs, Stefan

    2013-01-01

    Fluorescence microscopy of the localization and the spatial and temporal dynamics of specifically labelled proteins is an indispensable tool in cell biology. Besides fluorescent proteins as tags, tag-mediated labelling utilizing self-labelling proteins as the SNAP-, CLIP-, or the Halo-tag are widely used, flexible labelling systems relying on exogenously supplied fluorophores. Unfortunately, labelling of live budding yeast cells proved to be challenging with these approaches because of the limited accessibility of the cell interior to the dyes. In this study we developed a fast and reliable electroporation-based labelling protocol for living budding yeast cells expressing SNAP-, CLIP-, or Halo-tagged fusion proteins. For the Halo-tag, we demonstrate that it is crucial to use the 6′-carboxy isomers and not the 5′-carboxy isomers of important dyes to ensure cell viability. We report on a simple rule for the analysis of 1H NMR spectra to discriminate between 6′- and 5′-carboxy isomers of fluorescein and rhodamine derivatives. We demonstrate the usability of the labelling protocol by imaging yeast cells with STED super-resolution microscopy and dual colour live cell microscopy. The large number of available fluorophores for these self-labelling proteins and the simplicity of the protocol described here expands the available toolbox for the model organism Saccharomyces cerevisiae. PMID:24205303

  16. 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

  17. Electrostatically Stabilized Magnetic Nanoparticles - An Optimized Protocol to Label Murine T Cells for in vivo MRI.

    PubMed

    Wuerfel, Eva; Smyth, Maureen; Millward, Jason M; Schellenberger, Eyk; Glumm, Jana; Prozorovski, Timour; Aktas, Orhan; Schulze-Topphoff, Ulf; Schnorr, Jörg; Wagner, Susanne; Taupitz, Matthias; Infante-Duarte, Carmen; Wuerfel, Jens

    2011-01-01

    We present a novel highly efficient protocol to magnetically label T cells applying electrostatically stabilized very small superparamagnetic iron oxide particles (VSOP). Our long-term aim is to use magnetic resonance imaging (MRI) to investigate T cell dynamics in vivo during the course of neuroinflammatory disorders such as experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Encephalitogenic T cells were co-incubated with VSOP, or with protamine-complexed VSOP (VProt), respectively, at different conditions, optimizing concentrations and incubation times. Labeling efficacy was determined by atomic absorption spectrometry as well as histologically, and evaluated on a 7 T MR system. Furthermore, we investigated possible alterations of T cell physiology caused by the labeling procedure. T cell co-incubation with VSOP resulted in an efficient cellular iron uptake. T2 times of labeled cells dropped significantly, resulting in prominent hypointensity on T2*-weighted scans. Optimal labeling efficacy was achieved by VProt (1 mM Fe/ml, 8 h incubation; T2 time shortening of ∼80% compared to untreated cells). Although VSOP promoted T cell proliferation and altered the ratio of T cell subpopulations toward a CD4(+) phenotype, no effects on CD4 T cell proliferation or phenotypic stability were observed by labeling in vitro differentiated Th17 cells with VProt. Yet, high concentrations of intracellular iron oxide might induce alterations in T cell function, which should be considered in cell tagging studies. Moreover, we demonstrated that labeling of encephalitogenic T cells did not affect pathogenicity; labeled T cells were still capable of inducing EAE in susceptible recipient mice. PMID:22203815

  18. Small-molecule labeling of live cell surfaces for three-dimensional super-resolution microscopy.

    PubMed

    Lee, Marissa K; Rai, Prabin; Williams, Jarrod; Twieg, Robert J; Moerner, W E

    2014-10-01

    Precise imaging of the cell surface of fluorescently labeled bacteria requires super-resolution methods because the size-scale of these cells is on the order of the diffraction limit. In this work, we present a photocontrollable small-molecule rhodamine spirolactam emitter suitable for non-toxic and specific labeling of the outer surface of cells for three-dimensional (3D) super-resolution (SR) imaging. Conventional rhodamine spirolactams photoswitch to the emitting form with UV light; however, these wavelengths can damage cells. We extended photoswitching to visible wavelengths >400 nm by iterative synthesis and spectroscopic characterization to optimize the substitution on the spirolactam. Further, an N-hydroxysuccinimide-functionalized derivative enabled covalent labeling of amines on the surface of live Caulobacter crescentus cells. Resulting 3D SR reconstructions of the labeled cell surface reveal uniform and specific sampling with thousands of localizations per cell and excellent localization precision in x, y, and z. The distribution of cell stalk lengths (a sub-diffraction-sized cellular structure) was quantified for a mixed population of cells. Pulse-chase experiments identified sites of cell surface growth. Covalent labeling with the optimized rhodamine spirolactam label provides a general strategy to study the surfaces of living cells with high specificity and resolution down to 10-20 nm. PMID:25222297

  19. Small-Molecule Labeling of Live Cell Surfaces for Three-Dimensional Super-Resolution Microscopy

    PubMed Central

    2015-01-01

    Precise imaging of the cell surface of fluorescently labeled bacteria requires super-resolution methods because the size-scale of these cells is on the order of the diffraction limit. In this work, we present a photocontrollable small-molecule rhodamine spirolactam emitter suitable for non-toxic and specific labeling of the outer surface of cells for three-dimensional (3D) super-resolution (SR) imaging. Conventional rhodamine spirolactams photoswitch to the emitting form with UV light; however, these wavelengths can damage cells. We extended photoswitching to visible wavelengths >400 nm by iterative synthesis and spectroscopic characterization to optimize the substitution on the spirolactam. Further, an N-hydroxysuccinimide-functionalized derivative enabled covalent labeling of amines on the surface of live Caulobacter crescentus cells. Resulting 3D SR reconstructions of the labeled cell surface reveal uniform and specific sampling with thousands of localizations per cell and excellent localization precision in x, y, and z. The distribution of cell stalk lengths (a sub-diffraction-sized cellular structure) was quantified for a mixed population of cells. Pulse-chase experiments identified sites of cell surface growth. Covalent labeling with the optimized rhodamine spirolactam label provides a general strategy to study the surfaces of living cells with high specificity and resolution down to 10–20 nm. PMID:25222297

  20. 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

  1. 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

  2. MR Imaging Features of Gadofluorine-Labeled Matrix-Associated Stem Cell Implants in Cartilage Defects

    PubMed Central

    Do, Thuy; Sutton, Elizabeth J.; Baehner, Frederick; Horvai, Andrew; Sennino, Barbara; McDonald, Donald; Meier, Reinhard; Misselwitz, Bernd; Link, Thomas M.; Daldrup-Link, Heike E.

    2012-01-01

    Objectives The purpose of our study was to assess the chondrogenic potential and the MR signal effects of GadofluorineM-Cy labeled matrix associated stem cell implants (MASI) in pig knee specimen. Materials and Methods Human mesenchymal stem cells (hMSCs) were labeled with the micelle-based contrast agent GadofluorineM-Cy. Ferucarbotran-labeled hMSCs, non-labeled hMSCs and scaffold only served as controls. Chondrogenic differentiation was induced and gene expression and histologic evaluation were performed. The proportions of spindle-shaped vs. round cells of chondrogenic pellets were compared between experimental groups using the Fisher's exact test. Labeled and unlabeled hMSCs and chondrocytes in scaffolds were implanted into cartilage defects of porcine femoral condyles and underwent MR imaging with T1- and T2-weighted SE and GE sequences. Contrast-to-noise ratios (CNR) between implants and adjacent cartilage were determined and analyzed for significant differences between different experimental groups using the Kruskal-Wallis test. Significance was assigned for p<0.017, considering a Bonferroni correction for multiple comparisons. Results Collagen type II gene expression levels were not significantly different between different groups (p>0.017). However, hMSC differentiation into chondrocytes was superior for unlabeled and GadofluorineM-Cy-labeled cells compared with Ferucarbotran-labeled cells, as evidenced by a significantly higher proportion of spindle cells in chondrogenic pellets (p<0.05). GadofluorineM-Cy-labeled hMSCs and chondrocytes showed a positive signal effect on T1-weighted images and a negative signal effect on T2-weighted images while Ferucarbotran-labeled cells provided a negative signal effect on all sequences. CNR data for both GadofluorineM-Cy-labeled and Ferucarbotran-labeled hMSCs were significantly different compared to unlabeled control cells on T1-weighted SE and T2*-weighted MR images (p<0.017). Conclusion hMSCs can be labeled by

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

    PubMed

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

    1993-05-01

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

  4. Comparison of the labeling efficiency of BrdU, DiI and FISH labeling techniques in bone marrow stromal cells.

    PubMed

    Li, Na; Yang, Hui; Lu, Lingling; Duan, Chunli; Zhao, Chunli; Zhao, Huanying

    2008-06-18

    Cells are generally labeled during in vivo implantation studies enabling the cells to be traced. The relationship between the labeling efficiency and cellular proliferation after transplantation is critical for the interpretation of data obtained by detection of the signals on tissue sections. Here, we compare cellular labeling methods of rat marrow stromal cells that were labeled with 5-bromo-2-deoxyuridine (BrdU), 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and fluorescence in situ hybridization (FISH). Our data show that (i) BrdU uniformly labeled the nuclei, (ii) DiI-labeled cells had many dots or stained clear and uniform when a longer exposure time was used during detection and (iii) FISH labeled the cells with dots along the edges of the nuclei. The labeling efficiency was 94.1+/-8.6%, 97.6+/-3.4% and 90.5+/-3.0%, in BrdU, DiI- and FISH-labeled cells, respectively. After sub-culturing of labeled cells, the percentage of BrdU-positive cells was found to be 71.9+/-18.0% and 18.4+/-6.9%, after the first and second passages, respectively. The percentage of DiI-labeled cells detected depended on the exposure time: a long exposure time (>10 s) resulted in identification of 95.1+/-4.0% and 94.5+/-3.9% DiI-positive cells after the first and second sub-cultures, respectively. The percentage of FISH-positive cells was found to be 87.0+/-3.0% and 89.1+/-9.7%. The BrdU labeling signal quickly decreased over time. Thus, BrdU should only be used to temporarily label dividing cells. In contrast, our data indicate that DiI and FISH labeling may be used to steadily trace cells during in vivo experiments. To our knowledge, this is the first time that the effects of different labeling methods over time have been examined during a cell transplantation study. PMID:18468584

  5. 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

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

    PubMed

    Lövgren, J; Blomberg, K

    1994-07-12

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

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

    PubMed

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

    1994-07-12

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

  8. 111Indium labeling of hepatocytes for analysis of short-term biodistribution of transplanted cells.

    PubMed

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

    1994-03-01

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

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. Photosensitized labeling of a functional multidrug transporter in living drug-resistant tumor cells

    SciTech Connect

    Raviv, Y.; Pollard, H.B.; Bruggemann, E.P.; Pastan, I.; Gottesman, M.M. )

    1990-03-05

    A 170,000-Da glycoprotein (P170 multidrug transporter) becomes specifically labeled in multidrug-resistant human KB carcinoma cells by the photolabile lipophilic membrane probe 5-(125I)iodonaphthalene-1-azide ((125I)INA) when photoactivation of the probe is triggered by energy transfer from intracellular doxorubicin or rhodamine 123. In contrast, in drug-sensitive cells, drug-induced specific labeling of membrane proteins with (125I)INA was not observed. Instead, multiple membrane proteins became labeled in a nonspecific manner. This phenomenon of drug-induced specific labeling of P170 by (125I)INA is observed only in living cells, but not in purified membrane vesicles or lysed cells. It is generated by doxorubicin and rhodamine 123, drugs that are chromophores and to which the cells exhibit resistance; but it is not observed with other drugs or dyes. Verapamil, a calcium channel blocker which reverses resistance to doxorubicin, also abolishes doxorubicin-induced specific (125I)INA labeling of P170. These results reveal that a specific interaction between P170 and doxorubicin takes place in living cells and demonstrate that P170 is directly involved in the mechanism of drug resistance in vivo. They also provide a possible means to label functional domains in the multidrug transporter. The results demonstrate that photosensitized (125I)INA labeling is a technique which provides sufficient spatial and time resolution to detect specific intracellular interactions between chromophores and proteins in vivo.

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

    PubMed Central

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

    2015-01-01

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

  15. 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

  16. 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

  17. In vivo red blood cell compatibility testing using indium-113m tropolone-labeled red blood cells

    SciTech Connect

    Morrissey, G.J.; Gravelle, D.; Dietz, G.; Driedger, A.A.; King, M.; Cradduck, T.D.

    1988-05-01

    In vivo radionuclide crossmatch is a method for identifying compatible blood for transfusion when allo- or autoantibodies preclude the use of conventional crossmatching techniques. A technique for labeling small volumes of donor red blood cells with (/sup 113m/In)tropolone is reported. The use of /sup 113m/In minimizes the accumulation of background radioactivity and the radiation dose especially so when multiple crossmatches are performed. Labeling red cells with (/sup 113m/In)tropolone is faster and easier to perform than with other radionuclides. Consistently high labeling efficiencies are obtained and minimal /sup 113m/In activity elutes from the labeled red blood cells. A case study involving 22 crossmatches is presented to demonstrate the technique. The radiation dose equivalent from /sup 113m/In is significantly less than with other radionuclides that may be used to label red cells.

  18. 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.

  19. 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

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

    PubMed Central

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

    2009-01-01

    SUMMARY Lentiviral vectors (LVs) are capable of labeling a broad spectrum of cell types, achieving stable expression of transgenes. However, for in vivo studies, the duration of marker gene expression has been highly variable. We have developed a series of LVs harboring different promoters for expressing reporter gene in mouse cells. Long-term culture and colony formation of several LV-labeled mouse melanoma cells showed that promoters derived from mammalian house-keeping genes, especially those encoding RNA polymerase II (Pol2) and ferritin (FerH), provided the highest consistency for reporter expression. For in vivo studies, primary B16BL6 mouse melanoma were infected with LVs whose luciferase-GFP fusion gene (Luc/GFP) was driven by either Pol2 or FerH promoters. When transplanted into syngeneic C57BL/6 mice, Luc/GFP-labeled B16BL6 mouse melanoma cells can be monitored by bioluminescence imaging in vivo, and GFP-positive cells can be isolated from the tumors by FACS. Pol2-Luc/GFP labeling, while lower in activity, was more sustainable than FerH-Luc/GFP labeling in B16BL6 over consecutive passages into mice. We conclude that Pol-2-Luc/GFP labeling allows long-term in vivo monitoring and tumor cell isolation in immunocompetent mouse melanoma models. SIGNIFICANCE In this study we have developed and identified lentiviral vectors that allow labeled mouse melanoma cells to maintain long-term and consistent expression of a bifunctional luciferase-GFP marker gene, even in syngeneic mice with an intact immune function. This cell-labeling system can be used to build immunocompetent mouse melanoma models that permit both tumor monitoring and FACS-based tumor cell isolation from tissues, greatly facilitating the in vivo study of melanoma. PMID:19175523

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

    PubMed

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

    2009-06-01

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

  2. Iodine-131 labeled anti-CEA antibodies uptake by Huerthle cell carcinoma

    SciTech Connect

    Abdel-Nabi, H.; Hinkle, G.H.; Falko, J.M.; Kelly, D.; Olsen, J.O.; Martin, E.W. Jr.

    1985-10-01

    Localization of Huerthle cell cancer deposits in the lung with I-131 labeled anti-carcinoembryonic antigen (CEA) monoclonal antibody is described. This technique may prove useful if conventional scanning with I-131 sodium iodide for distant metastases is negative.

  3. Multicolor protein labeling in living cells using mutant β-lactamase-tag technology.

    PubMed

    Watanabe, Shuji; Mizukami, Shin; Hori, Yuichiro; Kikuchi, Kazuya

    2010-12-15

    Protein labeling techniques using small molecule probes have become important as practical alternatives to the use of fluorescent proteins (FPs) in live cell imaging. These labeling techniques can be applied to more sophisticated fluorescence imaging studies such as pulse-chase imaging. Previously, we reported a novel protein labeling system based on the combination of a mutant β-lactamase (BL-tag) with coumarin-derivatized probes and its application to specific protein labeling on cell membranes. In this paper, we demonstrated the broad applicability of our BL-tag technology to live cell imaging by the development of a series of fluorescence labeling probes for this technology, and the examination of the functions of target proteins. These new probes have a fluorescein or rhodamine chromophore, each of which provides enhanced photophysical properties relative to coumarins for the purpose of cellular imaging. These probes were used to specifically label the BL-tag protein and could be used with other small molecule fluorescent probes. Simultaneous labeling using our new probes with another protein labeling technology was found to be effective. In addition, it was also confirmed that this technology has a low interference with respect to the functions of target proteins in comparison to GFP. Highly specific and fast covalent labeling properties of this labeling technology is expected to provide robust tools for investigating protein functions in living cells, and future applications can be improved by combining the BL-tag technology with conventional imaging techniques. The combination of probe synthesis and molecular biology techniques provides the advantages of both techniques and can enable the design of experiments that cannot currently be performed using existing tools. PMID:20961132

  4. 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

  5. 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

  6. Study on the best method of SPIO labeling on the cell line ECV304

    NASA Astrophysics Data System (ADS)

    Yang, FuYuan; Yu, MingXi; Chen, WenLi; Zhou, Quan

    2010-02-01

    Studies the superparamagnetic iron oxide (SPIO) label ECV304 method and test its effect on cell physiological activity .We use different concentration PLL-SPIO incubate ECV304 cells, Prussian blue dye was taken to examine SPIO mark efficiency, DCF dyeing and PI & Annexin the VFITC double dyeing was carried out to analyze cell apoptosis. The result shows that 25μg/ml gave the most efficient labeling concentration, there is also no toxic effect on cells in this concentration. This outcome can be used in MRI of living body cell transplant tracking.

  7. 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

  8. Double labeling autoradiography. Cell kinetic studies with /sup 3/H- and /sup 14/C-thymidine

    SciTech Connect

    Schultze, B.

    1981-01-01

    Examples of the multiple applicability of the double labeling method with /sup 3/H- and /sup 14/C-TdR are demonstrated. Double labeling with /sup 3/H- and /sup 14/C-TdR makes it possible to determine the cycle and its phases with high precision by modifying the usual percent labeled mitoses method with a single injection of /sup 3/H-TdR. In addition, data is provided on the variances of the transit times through the cycle phases. For example, in the case of the jejunal crypt cells of the mouse, the transit times through successive cycle phases are uncorrelated. In the case of glial cells the double labeling method provides cell kinetic parameters despite the paucity of proliferating glial cells. In the adult untreated animal, glial cell mitoses are so rare that the percent labeled mitoses method can not be utilized. However, the S-phase duration can be measured by double labeling and the cycle time can be determined by the so-called method of labeled S phases. With the latter method the passage through the S phase of the /sup 3/H-TdR-labeled S phase cells can be registered by injecting /sup 14/C-TdR at different time intervals following /sup 3/H-TdR application. In this way an S-phase duration of about 10 hr and a cycle time of about 20 hr was found for glial cells in the adult untreated mouse. An exchange of glial cells between the growth fraction and the nongrowth fraction has also been shown by double labeling. A quite different application of the double labeling method with 3H- and /sup 14/C-TdR is the in vivo study of the cell cycle phase-specific effect of drugs used in chemotherapy of tumors. The effect of vincristine on these cells has been studied. Vincristine affects cells in S and G2 in such a manner that they are arrested during the next metaphase and subsequently become necrotic. It has no effect on G1 cells.

  9. Superparamagnetic iron oxide is suitable to label tendon stem cells and track them in vivo with MR imaging

    PubMed Central

    Yang, Yunfa; Zhang, Jianying; Qian, Yongxian; Dong, Shiwu; Huang, He; Boada, Fernando E; Fu, Freddie H.; Wang, James H-C.

    2013-01-01

    Tendon stem cells (TSCs) may be used to effectively repair or regenerate injured tendons. However, the fates of TSCs once implanted in vivo remain unclear. This study was aimed to determine the feasibility of labeling TSCs with super-paramagnetic iron oxide (SPIO) nano-particles to track TSCs in vivo using MRI. Rabbit TSCs were labeled by incubation with 50 μg/ml SPIO. Labeling efficiency, cell viability, and proliferation were then measured, and the stemness of TSCs was tested by quantitative real time RT-PCR (qRT-PCR) and immunocytochemistry. We found that the labeling efficiency of TSCs reached as high as 98%, and that labeling at 50 μg/ml SPIO concentrations did not alter cell viability and cell proliferation compared to non-labeled control cells. Moreover, the expression levels of stem cell markers (Nucleostemin, Nanog, and Oct-4) did not change in SPIO-labeled TSCs compared to non-labeled cells. Both labeled and non-labeled cells also exhibited similar differentiation potential. Finally, labeled TSCs could be detected by MRI both in vitro and in vivo. Taken together, the findings of this study show that labeling TSCs with SPIO particles is a feasible approach to track TSCs in vivo by MRI, which offers a noninvasive method to monitor repair of injured tendons. PMID:23549900

  10. 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

  11. 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...

  12. 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

  13. A covalent approach for site-specific RNA labeling in Mammalian cells.

    PubMed

    Li, Fahui; Dong, Jianshu; Hu, Xiaosong; Gong, Weimin; Li, Jiasong; Shen, Jing; Tian, Huifang; Wang, Jiangyun

    2015-04-01

    Advances in RNA research and RNA nanotechnology depend on the ability to manipulate and probe RNA with high precision through chemical approaches, both in vitro and in mammalian cells. However, covalent RNA labeling methods with scope and versatility comparable to those of current protein labeling strategies are underdeveloped. A method is reported for the site- and sequence-specific covalent labeling of RNAs in mammalian cells by using tRNA(Ile2) -agmatidine synthetase (Tias) and click chemistry. The crystal structure of Tias in complex with an azide-bearing agmatine analogue was solved to unravel the structural basis for Tias/substrate recognition. The unique RNA sequence specificity and plastic Tias/substrate recognition enable the site-specific transfer of azide/alkyne groups to an RNA molecule of interest in vitro and in mammalian cells. Subsequent click chemistry reactions facilitate the versatile labeling, functionalization, and visualization of target RNA. PMID:25694369

  14. 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

  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. PMID:27218228

  16. 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.

  17. Magnetic resonance tracking of nanoparticle labelled neural stem cells in a rat's spinal cord

    NASA Astrophysics Data System (ADS)

    Wang, F. H.; Lee, I. H.; Holmström, N.; Yoshitake, T.; Kim, D. K.; Muhammed, M.; Frisén, J.; Olson, L.; Spenger, C.; Kehr, J.

    2006-04-01

    Neural stem cells isolated from an adult rat's spinal cord were loaded with superparamagnetic gold-coated monocrystalline iron oxide nanoparticles (Au-MION) intended for use as contrast enhancers in magnetic resonance imaging (MRI). A dose-dependent attenuation of MRI signals was observed for Au-MION down to 0.001 µg Fe/µl and for nanoparticle-loaded clusters of only 20 cells. The labelled cells were infused into the spinal cord of anaesthetized rats and tracked by MRI at 1 h, 48 h and 1 month post-injection. Histological analysis revealed that MRI signals correlated well with gold-positive staining of transplanted cells. The present results show that Au-MION exerts powerful contrast-enhancing properties and may represent novel MRI labels for labelling and tracking the transplanted cells in vivo.

  18. 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.

  19. Labeling of stem cells with monocrystalline iron oxide for tracking and localization by magnetic resonance imaging

    PubMed Central

    Calzi, Sergio Li; Kent, David L.; Chang, Kyung-Hee; Padgett, Kyle R.; Afzal, Aqeela; Chandra, Saurav B.; Caballero, Sergio; English, Denis; Garlington, Wendy; Hiscott, Paul S.; Sheridan, Carl M.; Grant, Maria B.; Forder, John R.

    2013-01-01

    Precise localization of exogenously delivered stem cells is critical to our understanding of their reparative response. Our current inability to determine the exact location of small numbers of cells may hinder optimal development of these cells for clinical use. We describe a method using magnetic resonance imaging to track and localize small numbers of stem cells following transplantation. Endothelial progenitor cells (EPC) were labeled with monocrystalline iron oxide nanoparticles (MIONs) which neither adversely altered their viability nor their ability to migrate in vitro and allowed successful detection of limited numbers of these cells in muscle. MION-labeled stem cells were also injected into the vitreous cavity of mice undergoing the model of choroidal neovascularization, laser rupture of Bruch’s membrane. Migration of the MION-labeled cells from the injection site towards the laser burns was visualized by MRI. In conclusion, MION labeling of EPC provides a non-invasive means to define the location of small numbers of these cells. Localization of these cells following injection is critical to their optimization for therapy. PMID:19345699

  20. Labeling of stem cells with monocrystalline iron oxide for tracking and localization by magnetic resonance imaging.

    PubMed

    Li Calzi, Sergio; Kent, David L; Chang, Kyung-Hee; Padgett, Kyle R; Afzal, Aqeela; Chandra, Saurav B; Caballero, Sergio; English, Denis; Garlington, Wendy; Hiscott, Paul S; Sheridan, Carl M; Grant, Maria B; Forder, John R

    2009-06-01

    Precise localization of exogenously delivered stem cells is critical to our understanding of their reparative response. Our current inability to determine the exact location of small numbers of cells may hinder optimal development of these cells for clinical use. We describe a method using magnetic resonance imaging to track and localize small numbers of stem cells following transplantation. Endothelial progenitor cells (EPC) were labeled with monocrystalline iron oxide nanoparticles (MIONs) which neither adversely altered their viability nor their ability to migrate in vitro and allowed successful detection of limited numbers of these cells in muscle. MION-labeled stem cells were also injected into the vitreous cavity of mice undergoing the model of choroidal neovascularization, laser rupture of Bruch's membrane. Migration of the MION-labeled cells from the injection site towards the laser burns was visualized by MRI. In conclusion, MION labeling of EPC provides a non-invasive means to define the location of small numbers of these cells. Localization of these cells following injection is critical to their optimization for therapy. PMID:19345699

  1. 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

  2. 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

  3. Selective methyl labeling of eukaryotic membrane proteins using cell-free expression.

    PubMed

    Linser, Rasmus; Gelev, Vladimir; Hagn, Franz; Arthanari, Haribabu; Hyberts, Sven G; Wagner, Gerhard

    2014-08-13

    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

  4. 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.

  5. 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.

  6. 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

  7. Site-directed spin labeling of proteins for distance measurements in vitro and in cells.

    PubMed

    Roser, P; Schmidt, M J; Drescher, M; Summerer, D

    2016-06-15

    Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy allows studying the structure, dynamics, and interactions of proteins via distance measurements in the nanometer range. We here give an overview of available spin labels, the strategies for their introduction into proteins, and the associated potentials for protein structural studies in vitro and in the context of living cells. PMID:27181459

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

    PubMed

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

    2015-12-01

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

  9. Covalent affinity labeling, radioautography, and immunocytochemistry localize the glucocorticoid receptor in rat testicular Leydig cells

    SciTech Connect

    Stalker, A.; Hermo, L.; Antakly, T. )

    1989-12-01

    The presence and distribution of glucocorticoid receptors in the rat testis were examined by using 2 approaches: in vivo quantitative radioautography and immunocytochemistry. Radioautographic localization was made possible through the availability of a glucocorticoid receptor affinity label, dexamethasone 21-mesylate, which binds covalently to the glucocorticoid receptor, thereby preventing dissociation of the steroid-receptor complex. Adrenalectomized adult rats were injected with a tritiated (3H) form of this steroid into the testis and the tissue was processed for light-microscope radioautography. Silver grains were observed primarily over the Leydig cells of the interstitial space and to a lesser extent, over the cellular layers which make up the seminiferous epithelium, with no one cell type showing preferential labeling. To determine the specificity of the labeling, a 25- or 50-fold excess of unlabeled dexamethasone was injected simultaneously with the same dose of (3H)-dexamethasone 21-mesylate. In these control experiments, a marked reduction in label intensity was noted over the Leydig as well as tubular cells. Endocytic macrophages of the interstitium were non-specifically labeled, indicating uptake of the ligand possibly by fluid-phase endocytosis. A quantitative analysis of the label confirmed the presence of statistically significant numbers of specific binding sites for glucocorticoids in both Leydig cells and the cellular layers of the seminiferous epithelium; 86% of the label was found over Leydig cells, and only 14% over the cells of the seminiferous epithelium. These binding data were confirmed by light-microscope immunocytochemistry using a monoclonal antibody to the glucocorticoid receptor.

  10. Analyzing DNA Replication III: Antibody Labeling of Incorporated Bromodeoxyuridine (BrdU) in Tissues and Cells.

    PubMed

    Jackson, Dean; Cook, Peter R

    2008-01-01

    INTRODUCTIONThe number of cells traversing the cell cycle and the rate of progression through it provide important indices of cell growth and tumorigenicity. S-phase cells can also be identified by their high content of DNA polymerase and proliferating cell nuclear antigen, a component of the leading-strand polymerase. Although both these markers can be detected rapidly and conveniently using the appropriate antibodies, neither are found exclusively in S-phase cells. Immunolabeling after incorporation of modified DNA precursors (e.g., 5-bromodeoxyuridine [BrdU, bromodeoxyuridine]) allows more rapid and precise detection of cells in S-phase of the cell cycle. BrdU is phosphorylated by cells to give BrdUTP, and this precursor is incorporated into DNA instead of deoxythimidine triphosphate. In living cells, BrdU is incorporated into replication sites that can then be detected using fluorochrome or enzyme-coupled antibodies. Alternatively, DNA synthesis sites can be labeled at high resolution by incubating cells with analogs of the natural precursors of DNA. After fixation to preserve nuclear morphology, the DNA duplex is denatured to allow antibodies access to the BrdU. Cells labeled in this way either in vivo or in vitro display a few hundred discrete nuclear sites early in S-phase, with distinct patterns of DNA replication that are characteristic of different stages of S-phase. This protocol describes two commonly used methods of denaturation, as well as techniques for antibody labeling of mounted tissues and encapsulated cells. PMID:21356891

  11. 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

  12. 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

  13. 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

  14. 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

  15. Label-free Electrophysiological Cytometry for Stem Cell-Derived Cardiomyocyte Clusters

    PubMed Central

    Myers, Frank B.; Abilez, Oscar J.; Zarins, Christopher K.; Lee, Luke P.

    2012-01-01

    Stem cell therapies hold great promise for repairing tissues damaged due to disease or injury. However, a major obstacle facing this field is the difficulty in identifying cells of a desired phenotype from the heterogeneous population that arises during stem cell differentiation. Conventional fluorescence flow cytometry and magnetic cell purification require exogenous labeling of cell surface markers which can interfere with the performance of the cells of interest. Here, we describe a non-genetic, label-free cell cytometry method based on electrophysiological response to stimulus. As many of the cell types relevant for regenerative medicine are electrically-excitable (e.g. cardiomyocytes, neurons, smooth muscle cells), this technology is well-suited for identifying cells from heterogeneous stem cell progeny without the risk and expense associated with molecular labeling or genetic modification. Our label-free cell cytometer is capable of distinguishing clusters of undifferentiated human induced pluripotent stem cells (iPSC) from iPSC-derived cardiomyocyte (iPSC-CM) clusters. The system utilizes a microfluidic device with integrated electrodes for both electrical stimulation and recording of extracellular field potential (FP) signals from suspended cells in flow. The unique electrode configuration provides excellent rejection of field stimulus artifact while enabling sensitive detection of FPs with a noise floor of 2 μVrms. Cells are self-aligned to the recording electrodes via hydrodynamic flow focusing. Based on automated analysis of these extracellular signals, the system distinguishes cardiomyocytes from non-cardiomyocytes. This is an entirely new approach to cell cytometry, in which a cell’s functionality is assessed rather than its expression profile or physical characteristics. PMID:23207961

  16. 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.

  17. 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

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

    PubMed

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

    2016-11-01

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

  19. 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.

  20. Quantum dot labeling using positive charged peptides in human hematopoetic and mesenchymal stem cells.

    PubMed

    Ranjbarvaziri, Sarah; Kiani, Sahar; Akhlaghi, Aliasghar; Vosough, Ahmad; Baharvand, Hossein; Aghdami, Nasser

    2011-08-01

    Quantum dots (QDs), as new and promising fluorescent probes, hold great potential in long term non-invasive bio-imaging, however there are many uncovered issues regarding their competency. In the present study, different QDs (525, 585 and 800 nm) were used to label CD133, CD34, CD14 and mesenchymal stem cells (MSCs) using positively charged peptides. Results demonstrated highly efficient internalization with the possible involvement of macropinocytosis. As indicated by LDH release and the TUNEL assay, no measurable effects on cell viability were detected at a concentration of 10 nM. QDs did not have any deleterious effects on normal cell functionality where both labeled CD133(+) cells and MSCs remarkably differentiated along multiple lineages with the use of the colony forming assay and adipo/osteo induction, respectively. Our results regarding QD maintenance revealed that these nano-particles are not properly stable and various excretion times have been observed depending on particle size and cell type. In vitro co-culture system and transplantation of labeled cells to an animal model showed that QDs leaked out from labeled cells and the released nano-particles were able to re-enter adjacent cells over time. These data suggest that before any utilization of QDs in bio-imaging and related applications, an efficient intra-cellular delivery technique should be considered to preserve QDs for a prolonged time as well as eliminating their leakage. PMID:21549422

  1. 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

  2. In situ label-free cell viability assessment of nucleus pulposus tissue.

    PubMed

    Dittmar, Roman; van Dijk, Bart G M; van Zandvoort, Marc A M J; Ito, Keita

    2014-04-01

    Regenerative medicine approaches aiming at treating degenerating intervertebral discs, a major cause of back pain, are increasingly tested in ex-vivo disc explant models mimicking in-vivo conditions. For assessing the efficacy of regenerative therapies, cell viability is commonly measured requiring specific labels to stain cells. Here, we demonstrate and evaluate how cellular auto-fluorescence can be utilized to non-invasively assess viability in disc tissue in-situ using label-free two-photon microscopy. Live and dead bovine disc cells (0% and 100% cell viability) from the nucleus pulposus were seeded into collagen gels and auto-fluorescence was characterized. Subsequently, nucleus pulposus explants were cultured for 6 days in media with different glucose supplementation (0, 0.25, 0.5, and 1 g/L) to induce different degrees of cell death. Then, samples were split and viability was assessed using label-free two-photon microscopy and conventional staining. Results show that live and dead nucleus pulposus cells systematically emit auto-fluorescent light with distinct characteristics. Cell viability values obtained with label-free microscopy did not significantly differ from those acquired with staining. In summary, monitoring auto-fluorescence facilitates accurate cell viability assessment in nucleus tissue requiring no additional dyes. Thus, this technique may be suitable for pre-clinical testing of regenerative therapies in nucleus pulposus cultures. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:545-550, 2014. PMID:24391094

  3. Use of indium-111-labeled cells in measurement of cellular dynamics of experimental cardiac allograft rejection

    SciTech Connect

    Oluwole, S.; Wang, T.; Fawwaz, R.; Satake, K.; Nowygrod, R.; Reemtsma, K.; Hardy, M.A.

    1981-01-01

    This study evaluates the kinetics and utility of infused indium-111-labeled cells in detecting rejection in ACI to Lewis rat heart allografts. Syngeneic leukocytes, lymph node lymphocytes, and platelets were isolated and labeled with indium-111 (/sup 111/In) oxine, respectively, and were infused i.v. into Lewis rats carrying beating ACI or syngeneic hearts from post-transplant days 0 to 6. Recipients were imaged serially at 24 hr after infusion of labeled cells followed by excision of both native and transplanted hearts for direct isotope count. Labeled leukocytes accumulative progressively in the allograft with the scan becoming positive by post-transplant day 4. The ratio of allograft to native heart isotope counts rose from 1.25 on day 1 to 10.07 (P less than 0.0001) on day 7. The Lewis recipients infused with labeled lymphocytes showed a positive scan on days 6 and 7 whereas the allograft to native heart isotope count ratio rose from 0.97 on day 1 to 5.33 (P less than 0.001) on day 7. Recipients infused with /sup 111/In-labeled platelets showed a positive scan on days 5 to 7 and the allograft to native heart isotope count ratio rose sharply from 2.56 on day 4 to 16.98 (P less than 0.005) on day 7. Syngeneic heart grafts failed to demonstrate significant accumulation of any of the labeled cell population. These studies confirm the importance of nonlymphocytic cells in cellular rejection, evaluate the kinetics of graft invasion by the various cell types, and suggest that the techniques used afford a method for a safe and an early detection of allograft rejection.

  4. 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. PMID:24222537

  5. 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

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

    PubMed Central

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

    2013-01-01

    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. PMID:23853708

  7. 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.

  8. 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

  9. 40 CFR 600.304-12 - Fuel economy label-special requirements for hydrogen fuel cell vehicles.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... requirements for hydrogen fuel cell vehicles. 600.304-12 Section 600.304-12 Protection of Environment... MOTOR VEHICLES Fuel Economy Labeling § 600.304-12 Fuel economy label—special requirements for hydrogen fuel cell vehicles. Fuel economy labels for hydrogen fuel cell vehicles must meet the...

  10. 40 CFR 600.304-12 - Fuel economy label-special requirements for hydrogen fuel cell vehicles.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... requirements for hydrogen fuel cell vehicles. 600.304-12 Section 600.304-12 Protection of Environment... MOTOR VEHICLES Fuel Economy Labeling § 600.304-12 Fuel economy label—special requirements for hydrogen fuel cell vehicles. Fuel economy labels for hydrogen fuel cell vehicles must meet the...

  11. 40 CFR 600.304-12 - Fuel economy label-special requirements for hydrogen fuel cell vehicles.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... requirements for hydrogen fuel cell vehicles. 600.304-12 Section 600.304-12 Protection of Environment... MOTOR VEHICLES Fuel Economy Labeling § 600.304-12 Fuel economy label—special requirements for hydrogen fuel cell vehicles. Fuel economy labels for hydrogen fuel cell vehicles must meet the...

  12. 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

  13. Investigation of Relationships Between Transverse Relaxation Rate, Diffusion Coefficient, and Labeled Cell Concentration in Ischemic Rat Brain Using MRI

    PubMed Central

    Athiraman, Hemanthkumar; Jiang, Quan; Ding, Guang Liang; Zhang, Li; Zhang, Zheng Gang; Wang, Lei; Arbab, Ali S.; Li, Qingjiang; Panda, Swayam; Ledbetter, Karen; Rad, Ali M.; Chopp, Michael

    2009-01-01

    MRI has been used to evaluate labeled cell migration and distribution. However, quantitative determination of labeled cell concentration using MRI has not been systematically investigated. In the current study, we investigated the relationships between labeled cell concentration and MRI parameters of transverse relaxation rate, R2, and apparent diffusion coefficient (ADC), in vitro in phantoms and in vivo in rats after stroke. Significant correlations were detected between iron concentration or labeled cell concentration and MRI measurements of R2, ADC, and ADC×R2 in vitro. In contrast, in vivo labeled cell concentration did not significantly correlate with R2, ADC, and ADC×R2. A major factor for the absence of a significant correlation between labeled cell concentration and MRI measurements in vivo may be attributed to background effects of ischemic tissue. By correcting the background effects caused by ischemic damage, ΔR2 (difference in R2 values in the ischemic tissue with and without labeled cells) exhibited a significant correlation to labeled cell concentration. Our study suggests that MRI parameters have the potential to quantitatively determine labeled cell concentration in vivo. PMID:19107898

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

    PubMed

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

    2016-05-01

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

  15. Detection of Cell Proliferation in Spider Embryos Using BrdU Labeling.

    PubMed

    Prpic, Nikola-Michael; Schoppmeier, Michael; Damen, Wim G M

    2008-01-01

    INTRODUCTIONThe spider Cupiennius salei, commonly known as the American wandering spider, is particularly useful for embryological studies because of the availability of tools to study and manipulate its embryonic development. Cupiennius is used to study axis formation, segmentation, appendage development, neurogenesis, and silk production. These studies contribute to our understanding of the evolution of these processes, but they also help us to understand the origin and diversification of evolutionary novelties. Comparisons between spiders and insects can show the degree of conservation and divergence of developmental mechanisms during arthropod evolution. Any embryological feature conserved between spiders and insects is likely to represent an ancestral feature for arthropods. Comparative molecular embryological work in insects and spiders should eventually allow us to define a molecular archetype for the phylum Arthropoda. This will be a necessary cornerstone for comparing the different metazoan phyla, including chordates. This protocol describes the detection of proliferating cells in whole-mount Cupiennius embryos. When labeled nucleotides are introduced into mitotically dividing cells, these cells incorporate the labels into the newly synthesized DNA. Thus, only cells that have synthesized DNA after the addition of the label will be detected. This protocol uses 5-bromo-2'-deoxy-uridine (BrdU) as a label that is subsequently detected by immunocytochemistry. BrdU labeling is a relatively easy way to detect cells that have recently synthesized DNA. The main advantage of this technique is that the label accumulates over time and, by varying the incubation time before fixation, an increasingly cumulative picture of cell proliferation activity can be obtained. PMID:21356702

  16. 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

  17. 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

  18. 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

  19. Magnetic Resonance Detection of CD34+ Cells from Umbilical Cord Blood Using a 19F Label

    PubMed Central

    Duinhouwer, Lucia E.; van Rossum, Bernard J. M.; van Tiel, Sandra T.; van der Werf, Ramon M.; Doeswijk, Gabriela N.; Haeck, Joost C.; Rombouts, Elwin W. J. C.; ter Borg, Mariëtte N. D.; Kotek, Gyula; Braakman, Eric; Cornelissen, Jan J.; Bernsen, Monique R.

    2015-01-01

    Impaired homing and delayed recovery upon hematopoietic stem cell transplantation (HSCT) with hematopoietic stem cells (HSC) derived from umbilical cord blood (UCB) is a major problem. Tracking transplanted cells in vivo will be helpful to detect impaired homing at an early stage and allows early interventions to improve engraftment and outcome after transplantation. In this study, we show sufficient intracellular labeling of UCB-derived CD34+ cells, with 19F-containing PLGA nanoparticles which were detectable with both flow cytometry and magnetic resonance spectroscopy (MRS). In addition, labeled CD34+ cells maintain their capacity to proliferate and differentiate, which is pivotal for successful engraftment after transplantation in vivo. These results set the stage for in vivo tracking experiments, through which the homing efficiency of transplanted cells can be studied. PMID:26394043

  20. 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.

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

    PubMed

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

    2015-10-01

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

  2. Label-free impedance detection of cancer cells.

    PubMed

    Venkatanarayanan, Anita; Keyes, Tia E; Forster, Robert J

    2013-02-19

    Ovarian cancer cells, SKOV3, have been immobilized onto platinum microelectrodes using anti-EPCAM capture antibodies and detected with high sensitivity using electrochemical impedance. The change in impedance following cell capture is strongly dependent on the supporting electrolyte concentration. By controlling the concentration of Dulbecco's phosphate buffered saline (DPBS) electrolyte, the double layer thickness can be manipulated so that the interfacial electric field interacts with the bound cells, rather than simply decaying across the antibody capture layer. Significantly, the impedance changes markedly upon cell capture over the frequency range from 3 Hz to 90 kHz. For example, using an alternating-current (ac) amplitude of 25 mV, a frequency of 81.3 kHz, and an open circuit potential (OCP) as the direct-current (dc) voltage, a detection limit of 4 captured cells was achieved. Assuming an average cell radius of 5 μm, the linear dynamic range is from 4 captured cells to 650 ± 2 captured cells, which is approximately equivalent to fractional coverages from 0.1% to 29%. An equivalent circuit that models the impedance response of the cell capture is discussed. PMID:23331159

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

    PubMed Central

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

    2016-01-01

    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. PMID:26915695

  4. 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.

  5. 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

  6. [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

  7. 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).

  8. Sulforhodamine 101 selectively labels human astrocytoma cells in an animal model of glioblastoma.

    PubMed

    Georges, Joseph F; Martirosyan, Nikolay L; Eschbacher, Jennifer; Nichols, Joshua; Tissot, Maya; Preul, Mark C; Feuerstein, Burt; Anderson, Trent; Spetzler, Robert F; Nakaji, Peter

    2014-05-01

    Sulforhodamine 101 (SR101) is a useful tool for immediate staining of astrocytes. We hypothesized that if the selectivity of SR101was maintained in astrocytoma cells, it could prove useful for glioma research. Cultured astrocytoma cells and acute slices from orthotopic human glioma (n=9) and lymphoma (n=6) xenografts were incubated with SR101 and imaged with confocal microscopy. A subset of slices (n=18) were counter-immunostained with glial fibrillary acidic protein and CD20 for stereological assessment of SR101 co-localization. SR101 differentiated astrocytic tumor cells from lymphoma cells. In acute slices, SR101 labeled 86.50% (±1.86; p<0.0001) of astrocytoma cells and 2.19% (±0.47; p<0.0001) of lymphoma cells. SR101-labeled astrocytoma cells had a distinct morphology when compared with in vivo astrocytes. Immediate imaging of human astrocytoma cells in vitro and in ex vivo rodent xenograft tissue labeled with SR101 can identify astrocytic tumor cells and help visualize the tumor margin. These features are useful in studying astrocytoma in the laboratory and may have clinical applications. PMID:24666692

  9. 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

    2015-01-01

    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. PMID:26554870

  10. 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

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

    PubMed

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

    2011-05-01

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

  12. 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

  13. Osteogenic differentiation of GFP-labeled human umbilical cord blood derived mesenchymal stem cells after cryopreservation.

    PubMed

    Liu, Guangpeng; Ye, Xinhai; Zhu, Yuchang; Li, Yulin; Sun, Jian; Cui, Lei; Cao, Yilin

    2011-10-01

    The osteogenic capacity of human umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) has been demonstrated both in vitro and in vivo. Therefore, cell labeling and storage are becoming necessary for researching the potential therapeutic use of UCB-MSCs for bone tissue engineering. The aim of this study was to determine the effect of cryopreservation on the osteogenic differentiation of green fluorescent protein (GFP)-marked UCB-MSCs in vitro. MSCs were isolated from full-term human UCB, expanded, transfected with the GFP gene, and then cryopreserved in liquid nitrogen for 4 weeks. After thawing, cell surface antigen markers and osteogenic potential were analyzed, and the luminescence of these cells was observed by fluorescence microscopy. The results demonstrate that cryopreservation has no effect on the cell phenotype, GFP expression or osteogenic differentiation of UCB-MSCs, showing that cryopreserved GFP-labeled UCB-MSCs might be applied for bone tissue engineering. PMID:21684270

  14. Monitoring virus entry into living cells using DiD-labeled dengue virus particles.

    PubMed

    Ayala-Nuñez, Nilda V; Wilschut, Jan; Smit, Jolanda M

    2011-10-01

    A variety of approaches can be applied to investigate the multiple steps and interactions that occur during virus entry into the host cell. Single-virus tracking is a powerful real-time imaging technique that offers the possibility to monitor virus-cell binding, internalization, intracellular trafficking behavior, and the moment of membrane fusion of single virus particles in living cells. Here we describe the development and applications of a single-virus tracking assay based on the use of DiD-labeled dengue virus (DENV) in BS-C-1 cells. In addition - and using the same experimental setup - we present a binding and fusion assay that can be used to obtain a rapid insight into the relative extent of virus binding to the cell surface and membrane fusion. Details of virus labeling and characterization, microscopy setup, protocols, data analysis, and hints for troubleshooting are described throughout the paper. PMID:21855634

  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. Zebrabow: multispectral cell labeling for cell tracing and lineage analysis in zebrafish

    PubMed Central

    Pan, Y. Albert; Freundlich, Tom; Weissman, Tamily A.; Schoppik, David; Wang, X. Cindy; Zimmerman, Steve; Ciruna, Brian; Sanes, Joshua R.; Lichtman, Jeff W.; Schier, Alexander F.

    2013-01-01

    Advances in imaging and cell-labeling techniques have greatly enhanced our understanding of developmental and neurobiological processes. Among vertebrates, zebrafish is uniquely suited for in vivo imaging owing to its small size and optical translucency. However, distinguishing and following cells over extended time periods remains difficult. Previous studies have demonstrated that Cre recombinase-mediated recombination can lead to combinatorial expression of spectrally distinct fluorescent proteins (RFP, YFP and CFP) in neighboring cells, creating a ‘Brainbow’ of colors. The random combination of fluorescent proteins provides a way to distinguish adjacent cells, visualize cellular interactions and perform lineage analyses. Here, we describe Zebrabow (Zebrafish Brainbow) tools for in vivo multicolor imaging in zebrafish. First, we show that the broadly expressed ubi:Zebrabow line provides diverse color profiles that can be optimized by modulating Cre activity. Second, we find that colors are inherited equally among daughter cells and remain stable throughout embryonic and larval stages. Third, we show that UAS:Zebrabow lines can be used in combination with Gal4 to generate broad or tissue-specific expression patterns and facilitate tracing of axonal processes. Fourth, we demonstrate that Zebrabow can be used for long-term lineage analysis. Using the cornea as a model system, we provide evidence that embryonic corneal epithelial clones are replaced by large, wedge-shaped clones formed by centripetal expansion of cells from the peripheral cornea. The Zebrabow tool set presented here provides a resource for next-generation color-based anatomical and lineage analyses in zebrafish. PMID:23757414

  17. 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.

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

    PubMed Central

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

    2013-01-01

    Membrane proteins (MPs) mediate a variety of cellular responses to extracellular signals. While MPs are intensely studied for their values as disease biomarkers and therapeutic targets, in situ investigation of binding kinetics of MPs with their ligands has been a challenge. Traditional approaches isolate MPs and then study them ex situ, which does not accurately reflect their native structures and functions. We present here a label-free plasmonic microscopy method to map the local binding kinetics of MPs in their native environment. This new analytical method can perform simultaneous plasmonic and fluorescence imaging, thus making it possible to combine the strengths of both label-based and label-free techniques in one system. Using this method, we have determined the distribution of MPs on the surface of single cells, and the local binding kinetic constants of different MPs. Furthermore, we have studied the polarization of the MPs on the cell surface during chemotaxis. PMID:23000999

  19. Targeting Aldehyde Dehydrogenase: a Potential Approach for Cell labeling

    PubMed Central

    Vaidyanathan, Ganesan; Song, Haijing; Affleck, Donna; McDougald, Darryl L.; Storms, Robert W.; Zalutksy, Michael R.; Chin, Bennett B.

    2009-01-01

    Introduction To advance the science and clinical application of stem cell therapy, the availability of a highly sensitive, quantitative, and translational method for tracking stem cells would be invaluable. Because hematopoetic stem cells express high levels of the cytosolic enzyme aldehyde dehydrogenase-1A1 (ALDH1), we sought to develop an agent that is specific to ALDH1 and thus to cells expressing the enzyme. Such an agent might be also helpful in identifying tumors that are resistant to cyclophosphomide chemotherapy because ALDH1 is known to be responsible for this resistance. Methods We developed schemes for the synthesis of two 3radioiodinated aldehdyes—N-formylmethyl-5-[*I]iodopyridine-3-carboxamide ([*I]FMIC) and 4-diethylamino-3-[*I]iodobenzaldehyde ([*I]DEIBA)—at no-carrier-added levels from their respective tin precursors. These agents were evaluated using pure ALDH1 and tumor cells that expressed the enzyme. Results The average radiochemical yields for the synthesis [125I]FMIC and [125I]DEIBA were 70 ± 5% and 47 ± 14%, respectively. ALDH1 converted both compounds to respective acids suggesting their suitability as ALDH1 imaging agents. Although ability of ALDH1 within the cells to oxidize one of these substrates was shown, specific uptake in ALDH-expressing tumor cells could not be demonstrated. Conclusion To pursue this approach for ALDH1 imaging, radiolabeled aldehydes need to be designed such that, in addition to being good substrates for ALDH1, the cognate products should be sufficiently polar so as to be retained within the cells. PMID:19875048

  20. 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

  1. 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

  2. Cryo-imaging of fluorescently labeled single cells in a mouse

    NASA Astrophysics Data System (ADS)

    Steyer, Grant J.; Roy, Debashish; Salvado, Olivier; Stone, Meredith E.; Wilson, David L.

    2009-02-01

    We developed a cryo-imaging system to provide single-cell detection of fluorescently labeled cells in mouse, with particular applicability to stem cells and metastatic cancer. The Case cryoimaging system consists of a fluorescence microscope, robotic imaging positioner, customized cryostat, PC-based control system, and visualization/analysis software. The system alternates between sectioning (10-40 μm) and imaging, collecting color brightfield and fluorescent blockface image volumes >60GB. In mouse experiments, we imaged quantum-dot labeled stem cells, GFP-labeled cancer and stem cells, and cell-size fluorescent microspheres. To remove subsurface fluorescence, we used a simplified model of light-tissue interaction whereby the next image was scaled, blurred, and subtracted from the current image. We estimated scaling and blurring parameters by minimizing entropy of subtracted images. Tissue specific attenuation parameters were found [uT : heart (267 +/- 47.6 μm), liver (218 +/- 27.1 μm), brain (161 +/- 27.4 μm)] to be within the range of estimates in the literature. "Next image" processing removed subsurface fluorescence equally well across multiple tissues (brain, kidney, liver, adipose tissue, etc.), and analysis of 200 microsphere images in the brain gave 97+/-2% reduction of subsurface fluorescence. Fluorescent signals were determined to arise from single cells based upon geometric and integrated intensity measurements. Next image processing greatly improved axial resolution, enabled high quality 3D volume renderings, and improved enumeration of single cells with connected component analysis by up to 24%. Analysis of image volumes identified metastatic cancer sites, found homing of stem cells to injury sites, and showed microsphere distribution correlated with blood flow patterns. We developed and evaluated cryo-imaging to provide single-cell detection of fluorescently labeled cells in mouse. Our cryo-imaging system provides extreme (>60GB), micron

  3. 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.

  4. Identification of newborn cells by BrdU labeling and immunocytochemistry in vivo.

    PubMed

    Magavi, Sanjay S; Macklis, Jeffrey D

    2008-01-01

    Bromodeoxyuridine, variously abbreviated as BrdU, BudR, and BrdUrd, is a halogenated thymidine analog that is permanently integrated into the DNA of dividing cells during DNA synthesis in S phase. BrdU can be immunocytochemically detected in vitro and in vivo, allowing the identification of cells that were dividing the period of BrdU exposure. In vivo, it has been used to identify the "birthdate" of cells during development, to examine the fate of postnatally generated cells, and to label cells before transplantation, for subsequent identification. PMID:18369768

  5. Changes in immunoferritin labeling of Rickettsia tsutsugamushi after serial cultivation in 60Co-irradiated BHK cells.

    PubMed Central

    Rikihisa, Y; Rota, T; Lee, T H; MacDonald, A B; Ito, S

    1979-01-01

    The immunolabeling characteristics of Rickettsia tsutsugamushi (Gilliam strain) were examined by using a purified immunoglobulin G fraction of antibody to R. tsutsugamushi raised in rabbits. Formalin-fixed rickettsiae were reacted with this antibody and then with ferritin-conjugated goat anti-rabbit Fc antibody. R. tsutsugamushi cultivated in yolk sacs was used to raise antibody for this study. When rickettsiae in BHK-21 cells infected from yolk sac seed material were immunoferritin labeled, the binding of ferritin was found to be dense and uniform on the outer surface of the rickettsiae in disrupted host cells. Immunolabeling of purified suspensions of extracellular rickettsiae resulted in the uniform ferritin labeling of the microorganism. Aggregation of these rickettsiae by antibody appeared to depend upon the purity of the pellets. Immunoferritin labeling examined at high magnification revealed ferritin very close to the outer dense leaflet of the outer membrane. On some rickettsiae or on focal sites of others, the labelin; was several ferritin particles thick, suggesting the presence of a thick coating. The immunoferritin labeling of R. tsutsugamushi during successive serial passages in BHK-21 cells revealed decreased labeling with each passage, and by the 10th passage there was no detectable labeling. However, these rickettsiae inoculated back into yolk sacs regained their immunoferritin labeling. R. tsutsugamushi passed back into yolk sacs after four serial propagations in BHK-21 cells regained their labeling on the first passage in yolk sacs. However, rickettsiae from the 20th serial passage in BHK-21 cells required five passages in yolk sacs to reestablish their previous labeling affinity. Rickettsiae which did not label after 20 passages in BHK cells regained some of their labeling characteristics when sonicated. Antibody against rickettsiae cultivated in BHK-21 cells continued labeling rickettsiae even after 9 serial passages in BHK-21 cells. Images PMID

  6. Compatibility of Superparamagnetic Iron Oxide Nanoparticle Labeling for 1H MRI Cell Tracking with 31P MRS for Bioenergetic Measurements

    PubMed Central

    Zhang, Zhuoli; Hancock, Brynne; Leen, Stephanie; Ramaswamy, Sharan; Sollott, Steven J.; Boheler, Kenneth R.; Juhaszova, Magdalena; Lakatta, Edward G.; Spencer, Richard G.; Fishbein, Kenneth W.

    2011-01-01

    Labeling of cells with superparamagnetic iron oxide nanoparticles permits cell tracking by 1H MRI while 31P MRS allows non-invasive evaluation of cellular bioenergetics. We evaluated the compatibility of these two techniques by obtaining 31P NMR spectra of iron-labeled and unlabeled immobilized C2C12 myoblast cells in vitro. Broadened but usable 31P spectra were obtained, and peak area ratios of resonances corresponding to intracellular metabolites showed no significant differences between labeled and unlabeled cell populations. We conclude that 31P NMR spectra can be obtained from cells labeled with sufficient iron to permit visualization by 1H imaging protocols and that these spectra have sufficient quality to be used in assessing metabolic status. This result introduces the possibility of using localized 31P MRS to evaluate the viability of iron-labeled therapeutic cells as well as surrounding host tissue in vivo. PMID:20853523

  7. Analyzing DNA Replication I: Labeling Animals, Tissues, and Cells with Bromodeoxyuridine (BrdU).

    PubMed

    Jackson, Dean; Cook, Peter R

    2008-01-01

    INTRODUCTIONThe number of cells traversing the cell cycle and the rate of progression through it provide important indices of cell growth and tumorigenicity. S-phase cells can also be identified by their high content of DNA polymerase and proliferating cell nuclear antigen, a component of the leading-strand polymerase. Although both these markers can be detected rapidly and conveniently using the appropriate antibodies, neither are found exclusively in S-phase cells. Immunolabeling after incorporation of modified DNA precursors (e.g., 5-bromodeoxyuridine [BrdU, bromodeoxyuridine]) allows more rapid and precise detection of cells in S-phase of the cell cycle. BrdU is phosphorylated by cells to give BrdUTP, and this precursor is incorporated into DNA instead of deoxythimidine triphosphate. In living cells, BrdU is incorporated into replication sites that can then be detected using fluorochrome or enzyme-coupled antibodies. Alternatively, DNA synthesis sites can be labeled at high resolution by incubating cells with analogs of the natural precursors of DNA. The cells are then fixed and the incorporation sites are detected using fluorochrome- or enzyme-tagged antibodies. Cells labeled in this way either in vivo or in vitro display a few hundred discrete nuclear sites early in S-phase, with distinct patterns of DNA replication that are characteristic of different stages of S-phase. "Foci" labeled after very short incubations correspond with sites where many replicons are duplicated simultaneously within massive protein complexes. This protocol provides details for incorporating DNA precursors into tissues in whole animals in vivo, isolated tissue in vitro, and cultured cells. PMID:21356889

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

    PubMed Central

    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. PMID:26981077

  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. 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.

  11. Combining Perfluorocarbon and Superparamagnetic Iron-oxide Cell Labeling for Improved and Expanded Applications of Cellular MRI

    PubMed Central

    Hitchens, T. Kevin; Liu, Li; Foley, Lesley M.; Simplaceanu, Virgil; Ahrens, Eric T.; Ho, Chien

    2014-01-01

    Purpose The ability to detect the migration of cells in living organisms is fundamental in understanding biological processes and important for the development of novel cell-based therapies to treat disease. MRI can be used to detect the migration of cells labeled with superparamagnetic iron-oxide (SPIO) or perfluorocarbon (PFC) agents. In this study, we explored combining these two cell-labeling approaches to overcome current limitations and enable new applications for cellular MRI. Methods We characterized 19F-NMR relaxation properties of PFC-labeled cells in the presence of SPIO and imaged cells both ex vivo and in vivo in a rodent inflammation model to demonstrate selective visualization of cell populations. Results We show that with UTE3D, RARE and FLASH 19F images one can uniquely identify PFC-labeled cells, co-localized PFC- and SPIO-labeled cells, and PFC/SPIO co-labeled cells. Conclusion This new methodology has the ability to improve and expand applications of MRI cell tracking. Combining PFC and SPIO strategies can potentially provide a method to quench PFC signal transferred from dead cells to macrophages, thereby eliminating false positives. In addition, combining these techniques could also be used to track two cell types simultaneously and probe cell-cell proximity in vivo with MRI. PMID:24478194

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. Novel Positively Charged Nanoparticle Labeling for In Vivo Imaging of Adipose Tissue-Derived Stem Cells

    PubMed Central

    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

  17. Hippocampal Cajal-Retzius cells project to the entorhinal cortex: retrograde tracing and intracellular labelling studies.

    PubMed

    Ceranik, K; Deng, J; Heimrich, B; Lübke, J; Zhao, S; Förster, E; Frotscher, M

    1999-12-01

    Cajal-Retzius (CR) cells are characteristic horizontally orientated, early-generated transient neurons in the marginal zones of the neocortex and hippocampus that synthesize the extracellular matrix protein reelin. They have been implicated in the pathfinding of entorhino-hippocampal axons, but their role in this process remained unclear. Here we have studied the axonal projection of hippocampal CR cells. Following injection of the carbocyanine dye DiI into the entorhinal cortex of aldehyde-fixed rat embryos and young postnatal rats, neurons in the outer molecular layer of the dentate gyrus and stratum lacunosum-moleculare of the hippocampus proper with morphological characteristics of CR cells were retrogradely labelled. In a time course analysis, the first retrogradely labelled CR cells were observed on embryonic day 17. This projection of hippocampal CR cells to the entorhinal cortex was confirmed by retrograde tracing with Fast Blue in new-born rats and by intracellular biocytin filling of CR cells in acute slices from young postnatal rat hippocampus/entorhinal cortex and in entorhino-hippocampal slice cocultures using infrared videomicroscopy in combination with the patch-clamp technique. In double-labelling experiments CR cells were identified by their immunocytochemical staining for reelin or calretinin, and their interaction with entorhino-hippocampal axons labelled by anterograde tracers was analysed. Future studies need to investigate whether this early transient projection of hippocampal CR cells to the entorhinal cortex is used as a template by the entorhinal axons growing to their target layers in the hippocampus. PMID:10594654

  18. Kinetics of cell labeling and thymidine replacement after continuous infusion of halogenated pyrimidines in vivo

    SciTech Connect

    Rodriguez, R.; Ritter, M.A.; Fowler, J.F.; Kinsella, T.J. )

    1994-04-30

    The authors present experiments on an in vivo human tumor xenograft continuously exposed to a fixed serum concentration of halogenated pyrimidines so as to study the kinetics of cell labeling and thymidine replacement. Human colon tumor (HCT-116) cells were injected subcutaneously into nude mice. After 10 days, most animals (>90%) developed measurable tumor nodules with a volume doubling time of 5 [+-] 1 days. Once the tumors reached a cross-sectional area of 0.25-0.30 cm[sup 2], miniosmotic pumps were implanted to deliver a dose of 100 mg/kg/day of IdUrd (iododeoxyuridine) by continuous infusion. After an IdUrd exposure time of 1-7 days, blood and tumor tissue were collected. The steady state serum IdUrd concentration was 0.95 [+-] 0.1 [mu]M, which is a clinically relevant concentration for a prolonged continuous intravenous infusion. The tumor cell potential doubling time (T[sub pot]) was 25. The percent IdUrd thymidine replacement and the fraction of cells labeled followed exponential saturation kinetics with a halflife of 33 and 27 h, respectively. After 5 days of exposure, the thymidine replacement in tumor cells was 2.0% and the fraction of tumor cells labeled was 94%. Immunohistochemical staining of IdUrd labeled tumor tissues showed an exposure-dependent gradient of cellular labeling that was initially highest in regions close to blood vessels. After 4 days of exposure at 100 mg/kg/day, there was an increase in the fraction of cells in G[sub 0] + G[sub 1] and a decrease in the S phase population, suggesting a block between G[sub 1] and S phase. They conclude that the in vivo kinetics of IdUrd thymidine replacement and fraction of cells labeled after continuous exposure followed exponential saturation kinetics with a halflife of approximately the potential doubling time of the tumor cell population. Some form of prolonged, or briefly interrupted, continuous infusion should be considered for clinical administration. 48 refs., 5 figs.

  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. Detection of BrdU-label Retaining Cells in the Lacrimal Gland: Implications for Tissue Repair

    PubMed Central

    You, Samantha; Tariq, Ayesha; Kublin, Claire L.; Zoukhri, Driss

    2011-01-01

    The purpose of the present study was to determine if the lacrimal gland contains 5-bromo-2’-deoxyuridine (BrdU)-label retaining cells and if they are involved in tissue repair. Animals were pulsed daily with BrdU injections for 7 consecutive days. After a chase period of 2, 4, or 12 weeks, the animals were sacrificed and the lacrimal glands were removed and processed for BrdU immunostaining. In another series of experiments, the lacrimal glands of 12-week chased animals were either left untreated or were injected with interleukin 1 (IL-1) to induce injury. Two and half day post-injection, the lacrimal glands were removed and processed for BrdU immunostaining. After 2 and 4 week of chase period, a substantial number of lacrimal gland cells were BrdU+ (11.98 ± 1.84 and 7.95 ± 1.83 BrdU+ cells/mm2, respectively). After 12 weeks of chase, there was a 97% decline in the number of BrdU+ cells (0.38 ± 0.06 BrdU+ cells/mm2), suggesting that these BrdU-label retaining cells may represent slow-cycling adult stem/progenitor cells. In support of this hypothesis, the number of BrdU labeled cells increased over 7-fold during repair of the lacrimal gland (control: 0.41 ± 0.09 BrdU+ cells/mm2, injured: 2.91 ± 0.62 BrdU+ cells/mm2). Furthermore, during repair, among BrdU+ cells 58.2 ± 3.6 % were acinar cells, 26.4 ± 4.1% were myoepithelial cells, 0.4 ± 0.4% were ductal cells, and 15.0 ± 3.0% were stromal cells. We conclude that the murine lacrimal gland contains BrdU-label retaining cells that are mobilized following injury to generate acinar, myoepithelial and ductal cells. PMID:22101331

  1. Detection of BrdU-label retaining cells in the lacrimal gland: implications for tissue repair.

    PubMed

    You, Samantha; Tariq, Ayesha; Kublin, Claire L; Zoukhri, Driss

    2011-12-01

    The purpose of the present study was to determine if the lacrimal gland contains 5-bromo-2'-deoxyuridine (BrdU)-label retaining cells and if they are involved in tissue repair. Animals were pulsed daily with BrdU injections for 7 consecutive days. After a chase period of 2, 4, or 12 weeks, the animals were sacrificed and the lacrimal glands were removed and processed for BrdU immunostaining. In another series of experiments, the lacrimal glands of 12-week chased animals were either left untreated or were injected with interleukin 1 (IL-1) to induce injury. Two and half days post-injection, the lacrimal glands were removed and processed for BrdU immunostaining. After 2 and 4 weeks of chase period, a substantial number of lacrimal gland cells were BrdU(+) (11.98 ± 1.84 and 7.95 ± 1.83 BrdU(+) cells/mm(2), respectively). After 12 weeks of chase, there was a 97% decline in the number of BrdU(+) cells (0.38 ± 0.06 BrdU(+) cells/mm(2)), suggesting that these BrdU-label retaining cells may represent slow-cycling adult stem/progenitor cells. In support of this hypothesis, the number of BrdU labeled cells increased over 7-fold during repair of the lacrimal gland (control: 0.41 ± 0.09 BrdU(+) cells/mm(2); injured: 2.91 ± 0.62 BrdU(+) cells/mm(2)). Furthermore, during repair, among BrdU(+) cells 58.2 ± 3.6 % were acinar cells, 26.4 ± 4.1% were myoepithelial cells, 0.4 ± 0.4% were ductal cells and 15.0 ± 3.0% were stromal cells. We conclude that the murine lacrimal gland contains BrdU-label retaining cells that are mobilized following injury to generate acinar, myoepithelial and ductal cells. PMID:22101331

  2. A Selective and Purification-Free Strategy for Labeling Adherent Cells with Inorganic Nanoparticles.

    PubMed

    Gao, Yu; Lim, Jing; Yeo, David Chen Loong; Liao, Shanshan; Lans, Malin; Wang, Yaqi; Teoh, Swee-Hin; Goh, Bee Tin; Xu, Chenjie

    2016-03-01

    Cellular labeling with inorganic nanoparticles such as magnetic iron oxide nanoparticles, quantum dots, and fluorescent silica nanoparticles is an important method for the noninvasive visualization of cells using various imaging modalities. Currently, this is mainly achieved through the incubation of cultured cells with the nanoparticles that eventually reach the intracellular compartment through specific or nonspecific internalization. This classic method is advantageous in terms of simplicity and convenience, but it suffers from issues such as difficulties in fully removing free nanoparticles (suspended in solution) and the lack of selectivity on cell types. This article reports an innovative strategy for the specific labeling of adherent cells without the concern of freely suspended nanoparticles. This method relies on a nanocomposite film that is prepared by homogeneously dispersing nanoparticles within a biodegradable polymeric film. When adherent cells are seeded on the film, they adhere, spread, and filtrate into the film through the micropores formed during the film fabrication. The pre-embedded nanoparticles are thus internalized by the cells during this infiltration process. As an example, fluorescent silica nanoparticles were homogeneously distributed within a polycaprolactone film by utilizing cryomilling and heat pressing. Upon incubation within physiological buffer, no silica nanoparticles were released from the nanocomposite film even after 20 d of incubation. However, when adherent cells (e.g., human mesenchymal stem cells) were grown on the film, they became fluorescent after 3 d, which suggests internalization of silica nanoparticles by cells. In comparison, the suspension cells (e.g., monocytes) in the medium remained nonfluorescent no matter whether there was the presence of adherent cells or not. This strategy eventually allowed the selective and concomitant labeling of mesenchymal stem cells during their harvest from bone marrow aspiration

  3. 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.

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

    PubMed Central

    Müller, Anke; Stellmacher, Anne; Freitag, Christine E.; Landgraf, Peter; Dieterich, Daniela C.

    2015-01-01

    The ability of the nervous system to undergo long-term plasticity is based on changes in cellular and synaptic proteomes. While many studies have explored dynamic alterations in neuronal proteomes during plasticity, there has been less attention paid to the astrocytic counterpart. Indeed, progress in identifying cell type-specific proteomes is limited owing to technical difficulties. Here, we present a cell type-specific metabolic tagging technique for a mammalian coculture model based on the bioorthogonal amino acid azidonorleucine and the mutated Mus musculus methionyl-tRNA synthetaseL274G enabling azidonorleucine introduction into de novo synthesized proteins. Azidonorleucine incorporation resulted in cell type-specific protein labeling and retained neuronal or astrocytic cell viability. Furthermore, we were able to label astrocytic de novo synthesized proteins and identified both Connexin-43 and 60S ribosomal protein L10a upregulated upon treatment with Brain-derived neurotrophic factor in astrocytes of a neuron-glia coculture. Taken together, we demonstrate the successful dissociation of astrocytic from neuronal proteomes by cell type-specific metabolic labeling offering new possibilities for the analyses of cell type-specific proteome dynamics. PMID:26690742

  5. 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.

  6. 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

  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. PMID:26153152

  8. 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.

  9. 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.

  10. 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.

  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. 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

  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. Electrostatically Stabilized Magnetic Nanoparticles – An Optimized Protocol to Label Murine T Cells for in vivo MRI

    PubMed Central

    Wuerfel, Eva; Smyth, Maureen; Millward, Jason M.; Schellenberger, Eyk; Glumm, Jana; Prozorovski, Timour; Aktas, Orhan; Schulze-Topphoff, Ulf; Schnorr, Jörg; Wagner, Susanne; Taupitz, Matthias; Infante-Duarte, Carmen; Wuerfel, Jens

    2011-01-01

    We present a novel highly efficient protocol to magnetically label T cells applying electrostatically stabilized very small superparamagnetic iron oxide particles (VSOP). Our long-term aim is to use magnetic resonance imaging (MRI) to investigate T cell dynamics in vivo during the course of neuroinflammatory disorders such as experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Encephalitogenic T cells were co-incubated with VSOP, or with protamine-complexed VSOP (VProt), respectively, at different conditions, optimizing concentrations and incubation times. Labeling efficacy was determined by atomic absorption spectrometry as well as histologically, and evaluated on a 7 T MR system. Furthermore, we investigated possible alterations of T cell physiology caused by the labeling procedure. T cell co-incubation with VSOP resulted in an efficient cellular iron uptake. T2 times of labeled cells dropped significantly, resulting in prominent hypointensity on T2*-weighted scans. Optimal labeling efficacy was achieved by VProt (1 mM Fe/ml, 8 h incubation; T2 time shortening of ∼80% compared to untreated cells). Although VSOP promoted T cell proliferation and altered the ratio of T cell subpopulations toward a CD4+ phenotype, no effects on CD4 T cell proliferation or phenotypic stability were observed by labeling in vitro differentiated Th17 cells with VProt. Yet, high concentrations of intracellular iron oxide might induce alterations in T cell function, which should be considered in cell tagging studies. Moreover, we demonstrated that labeling of encephalitogenic T cells did not affect pathogenicity; labeled T cells were still capable of inducing EAE in susceptible recipient mice. PMID:22203815

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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

  1. Resonant waveguide grating biosensor-enabled label-free and fluorescence detection of cell adhesion

    PubMed Central

    Zaytseva, Natalya; Lynn, Jeffery G.; Wu, Qi; Mudaliar, Deepti J.; Sun, Haiyan; Kuang, Patty Q.; Fang, Ye

    2013-01-01

    Cell adhesion to extracellular matrix (ECM) is fundamental to many distinct aspects of cell biology, and has been an active topic for label-free biosensors. However, little attention has been paid to study the impact of receptor signaling on the cell adhesion process. We here report the development of resonant waveguide grating biosensor-enabled label-free and fluorescent approaches, and their use for investigating the adhesion of an engineered HEK-293 cell line stably expressing green fluorescent protein (GFP) tagged β2-adrenergic receptor (β2-AR) onto distinct surfaces under both ambient and physiological conditions. Results showed that cell adhesion is sensitive to both temperature and ECM coating, and distinct mechanisms govern the cell adhesion process under different conditions. The β2-AR agonists, but not its antagonists or partial agonists, were found to be capable of triggering signaling during the adhesion process, leading to an increase in the adhesion of the engineered cells onto fibronectin-coated biosensor surfaces. These results suggest that the dual approach presented is useful to investigate the mechanism of cell adhesion, and to identify drug molecules and receptor signaling that interfere with cell adhesion. PMID:24319319

  2. Differentiation capacity of BrdU label-retaining dental pulp cells during pulpal healing following allogenic transplantation in mice.

    PubMed

    Saito, Kotaro; Ishikawa, Yuko; Nakakura-Ohshima, Kuniko; Ida-Yonemochi, Hiroko; Nakatomi, Mitsushiro; Kenmotsu, Shin-Ichi; Ohshima, Hayato

    2011-08-01

    Our recent study has demonstrated the localization of putative dental pulp stem cells in the developing molar by chasing 5-bromo-2'-deoxyuridine (BrdU)-labeling. However, their differentiation capacity subsequent to the tooth transplantation remains to be elucidated. This study aims to clarify the differentiation capacity of BrdU label-retaining dental pulp cells and their relationship to cell proliferation and apoptosis during pulpal healing following allogenic transplantation in mice. Following extraction of the mouse molar in BrdU-labeled animals, the roots and pulp floor were resected and immediately allo-grafted into the sublingual region in non-labeled animals, and vice versa. In the labeled transplants, label-retaining cells (LRCs) were increased in number and committed in nestin-positive newly differentiated odontoblast-like cells, whereas they were not committed in osteoblast-like cells. In the labeled host, on the contrary, LRCs were committed in neither odontoblast- nor osteoblast-like cells, although they were transiently increased in number and finally disappeared in the pulp tissue of the transplants. Interestingly, numerous apoptotic cells appeared in the pulp tissue including LRCs during the experimental period. These results suggest that transplanted LRCs maintain their proliferative and differentiation capacity in spite of extensive apoptosis occurring in the transplant, whereas transiently increased host-derived LRCs finally disappear in the pulp chamber following apoptosis. PMID:21878732

  3. Gene-specific cell labeling using MiMIC transposons

    PubMed Central

    Gnerer, Joshua P.; Venken, Koen J. T.; Dierick, Herman A.

    2015-01-01

    Binary expression systems such as GAL4/UAS, LexA/LexAop and QF/QUAS have greatly enhanced the power of Drosophila as a model organism by allowing spatio-temporal manipulation of gene function as well as cell and neural circuit function. Tissue-specific expression of these heterologous transcription factors relies on random transposon integration near enhancers or promoters that drive the binary transcription factor embedded in the transposon. Alternatively, gene-specific promoter elements are directly fused to the binary factor within the transposon followed by random or site-specific integration. However, such insertions do not consistently recapitulate endogenous expression. We used Minos-Mediated Integration Cassette (MiMIC) transposons to convert host loci into reliable gene-specific binary effectors. MiMIC transposons allow recombinase-mediated cassette exchange to modify the transposon content. We developed novel exchange cassettes to convert coding intronic MiMIC insertions into gene-specific binary factor protein-traps. In addition, we expanded the set of binary factor exchange cassettes available for non-coding intronic MiMIC insertions. We show that binary factor conversions of different insertions in the same locus have indistinguishable expression patterns, suggesting that they reliably reflect endogenous gene expression. We show the efficacy and broad applicability of these new tools by dissecting the cellular expression patterns of the Drosophila serotonin receptor gene family. PMID:25712101

  4. 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. PMID:23789985

  5. 19F MRI detection of acute allograft rejection with in vivo perfluorocarbon labeling of immune cells.

    PubMed

    Hitchens, T Kevin; Ye, Qing; Eytan, Danielle F; Janjic, Jelena M; Ahrens, Eric T; Ho, Chien

    2011-04-01

    Current diagnosis of organ rejection following transplantation relies on tissue biopsy, which is not ideal due to sampling limitations and risks associated with the invasive procedure.We have previously shown that cellular magnetic resonance imaging (MRI) of iron-oxide labeled immune-cell infiltration can provide a noninvasive measure of rejection status by detecting areas of hypointensity on T 2*-weighted images. In this study, we tested the feasibility of using a fluorine-based cellular tracer agent to detect macrophage accumulation in rodent models of acute allograft rejection by fluorine-19 ((19) F) MRI and magnetic resonance spectroscopy. This study used two rat models of acute rejection, including abdominal heterotopic cardiac transplant and orthotopic kidney transplant models. Following in vivo labeling of monocytes and macrophages with a commercially available agent containing perfluoro-15-crown-5-ether, we observed (19) F-signal intensity in the organs experiencing rejection by (19) F MRI, and conventional (1) H MRI was used for anatomical context. Immunofluorescence and histology confirmed macrophage labeling. These results are consistent with our previous studies and show the complementary nature of the two cellular imaging techniques. With no background signal, (19) F MRI/magnetic resonance spectroscopy can provide unambiguous detection of fluorine labeled cells, and may be a useful technique for detecting and quantifying rejection grade in patients. PMID:21305593

  6. DNA and chromosome breaks induced by iodine-123-labeled estrogen in Chinese hamster ovary cells

    SciTech Connect

    Schwartz, J.L. |; Mustafi, R.; Hughes, A.; DeSombre, E.R.

    1996-08-01

    The effects of the Auger electron-emitting isotope {sup 123}I, covalently bound to estrogen, on DNA single- and double-strand breakage and on chromosome breakage was determined in estrogen receptor-positive Chinese hamster ovary (CHO-ER) cells. Exposure to the {sup 123}I-labeled estrogen induced both single- and double-strand breaks with a ratio of single- to double-strand breaks of 2.8. The corresponding ratio with {sup 60}Co {gamma} rays was 15.6. The dose response was biphasic, suggesting either that receptor sites are saturated at high doses, or that there is a nonrandom distribution of breaks induced by the {sup 123}I-labeled estrogen. The {sup 123}I-labeled estrogen treatment induced chromosome aberrations with an efficiency of about 1 aberration for each 1000 disintegrations per cell. This corresponds to the mean lethal dose of {sup 123}I-labeled estrogen for these cells, suggesting that the lethal event induced by the Auger electron emitter bound to estrogen is a chromosome aberration. Most of the chromosome-type aberrations were dicentrics and rings, suggesting that {sup 123}I-labeled estrogen-induced chromosome breaks are rejoined. The F ratio, the ratio of dicentrics to centric rings, was 5.8 {+-} 1.7, which is similar to that seen with high-LET radiations. Our results suggest that {sup 123}I bound to estrogen is an efficient clastogenic agent, the cytotoxic damage produced by {sup 123}I bound to estrogen is very like damage induced by high-LET radiation, and the {sup 123}I in the estrogen receptor-DNA complex is probably in proximity to the sugar-phosphate backbone of the DNA. 40 refs., 7 figs.

  7. 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

  8. 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.

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

    PubMed Central

    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.

    2015-01-01

    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-coenzyme A 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 media with commercially available [13C3 15N1]-pantothenic acid, mammalian cells exclusively incorporated [13C3 15N1]-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 [13C3 15N1]-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. PMID:25572876

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. Suitability of Cell-Based Label-Free Detection for Cytotoxicity Screening of Carbon Nanotubes

    PubMed Central

    Meindl, Claudia; Absenger, Markus; Roblegg, Eva; Fröhlich, Eleonore

    2013-01-01

    Cytotoxicity testing of nanoparticles (NPs) by conventional screening assays is often complicated by interference. Carbon nanotubes (CNTs) are particularly difficult to assess. To test the suitability of cell-based label-free techniques for this application, a panel of CNTs with different diameters and surface functionalizations was assessed by impedance-based technique (xCELLigence RTCA) and automated microscopy (Cell-IQ) compared to formazan bioreduction (MTS assay). For validation of the label-free systems different concentrations of ethanol and of amine (AMI) polystyrene NPs were used. CNTs were evaluated in various cell lines, but only endothelial EAhy926 cells and L929 and V79 fibroblasts could be evaluated in all systems. Polystyrene particles obtained similar results in all assays. All systems identified thin (<8 nm) CNTs as more cytotoxic than thick (>20 nm) CNTs, but detection by xCELLigence system was less sensitive to CNT-induced cytotoxicity. Despite advantages, such as continuous monitoring and more detailed analysis of cytotoxic effects, label-free techniques cannot be generally recommended for cytotoxicity screening of NPs. PMID:24377092

  17. Sustained Analgesic Peptide Secretion and Cell Labeling Using a Novel Genetic Modification

    PubMed Central

    Gajavelli, Shyam; Castellanos, Daniel A.; Furmanski, Orion; Schiller, Paul C.; Sagen, Jacqueline

    2009-01-01

    Cell-based therapy for neuropathic pain could provide analgesics to local pain modulatory regions in a sustained, renewable fashion. In order to provide enhanced analgesic efficacy, transplantable cells may be engineered to produce complementary or increased levels of analgesic peptides. In addition, genetic labeling of modified cells is desirable for identification and tracking, but it should be retained intracellularly as desired analgesic peptides are secreted. Usually constructs encode proteins destined for either extra- or intra-cellular compartments, as these pathways do not cross. However, interactions between intracellular destinations provide a window of opportunity to overcome this limitation. In this report, we have explored this approach using a potential supplementary analgesic peptide, [Ser1]-histogranin (SHG), the stable synthetic derivative of a naturally occurring peptide with N-methyl D-aspartate (NMDA) antagonistic properties. A synthetic SHG gene was combined with (i) nerve growth factor-β (NGF-β) amino-terminal signal peptide to enable secretion, and (ii) a fluorescent cellular label (mRFP) with intervening cathepsin L cleavage site, and subcloned into a lentiviral vector. In addition, an endoplasmic retention signal, KDEL, was added to enable retrieval of mRFP. Using immunocytochemistry and confocal microscopic profile analysis, cells transduced by such lentiviruses were shown to synthesize a single SHG-mRFP polypeptide that was processed, targeted to expected subcellular destinations in several cell types. Dot blot and Western analysis revealed stable transduction and long-term secretion of SHG from PC12 cells in vitro. Transplantation of such cells provided modest analgesia in a rodent pain model consistent with low levels of SHG peptide in the cerebrospinal fluid (CSF). These results suggest that it is possible to deliver proteins with different final destinations from a single construct, such as pharmacologically active peptide for

  18. Frequency-selective REDOR and spin-diffusion relays in uniformly labeled whole cells.

    PubMed

    Rice, David M; Romaniuk, Joseph A H; Cegelski, Lynette

    2015-11-01

    Solid-state NMR is a powerful and non-perturbative method to measure and define chemical composition and architecture in bacterial cell walls, even in the context of whole cells. Most NMR studies on whole cells have used selectively labeled samples. Here, we introduce an NMR sequence relay using frequency-selective REDOR (fsREDOR) and spin diffusion elements to probe a unique amine contribution in uniformly (13)C- and (15)N-labeled Staphylococcus aureus whole cells that we attribute to the d-alanine of teichoic acid. In addition to the primary peptidoglycan structural scaffold, cell walls can contain significant amounts of teichoic acid that contribute to cell-wall function. When incorporated into teichoic acid, d-alanine is present as an ester, connected via its carbonyl to a ribitol carbon, and thus has a free amine. Teichoic acid d-Ala is removed during cell-wall isolations and can only be detected in the context of whole cells. The sequence presented here begins with fsREDOR and a chemical shift evolution period for 2D data acquisition, followed by DARR spin diffusion and then an additional fsREDOR period. fsREDOR elements were used for (13)C observation to avoid complications from (13)C-(13)C couplings due to uniform labeling and for (15)N dephasing to achieve selectivity in the nitrogens serving as dephasers. The results show that the selected amine nitrogen of interest is near to teichoic acid ribitol carbons and also the methyl group carbon associated with alanine. In addition, its carbonyl is not significantly dephased by amide nitrogens, consistent with the expected microenvironment around teichoic acid. PMID:26493462

  19. 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

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

    NASA Astrophysics Data System (ADS)

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

    2010-02-01

    For description of cellular phenotypes and physiological states new developments are needed. Axetris' impedance flow cytometer (IFC) (Leister) is a new promising label-free alternative to fluorescence-based flow cytometry (FCM). IFC measures single cells at various frequencies simultaneously. The frequencies used for signal acquisition range from 0.1 to 20 MHz. The impedance signal provides information about cell volume (< 1 MHz), membrane capacitance (~1-4 MHz) and cytoplasmic conductivity (4-10 MHz), parameters directly related to the physiological conditions of single cells. In MCF-7 cell viability experiments, cells were treated with cytotoxic agents to induce cell death. Impedance analysis showed discrimination between viable and dead cells. This was clearly visible at 4 MHz suggesting that differentiation was possible based on cell membrane capacitance. Changes in cell membrane potential were also analysed by IFC. RN22 cells were loaded with membrane potential sensitive dye (DiBAC4). The cells were then treated with the ionophore valinomycin. Changes in membrane potential were detectable at the level of cytoplasm conductivity (>4 MHz) and membrane capacitance (1-4 MHz). Our data indicate that IFC can be a valuable alternative to conventional FCM for various applications in the field of cell death and physiology. The work will be extended to address further potential applications of IFC in biotechnology and biomedical cell analysis, as well as in cell sorting.

  1. 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.

  2. A new metabolic cell wall labeling method reveals peptidoglycan in Chlamydia trachomatis

    PubMed Central

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

    2014-01-01

    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 pressure1. Bacteria comprising the Chlamydiales were thought to be one of the few exceptions. Chlamydia encodes genes for PG biosynthesis2–7 and exhibits susceptibility to "anti-PG" antibiotics8,9, yet attempts to detect PG in any chlamydial species have proven unsuccessful (the ‘chlamydial anomaly’10). We employed a novel approach to metabolically label chlamydial PG using D-amino acid dipeptide probes and click chemistry. Replicating Chlamydia trachomatis was labeled with the probes throughout its biphasic, developmental life cycle, and differential probe incorporation experiments conducted in the presence of ampicillin is 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 to date that chlamydial species possess functional PG. PMID:24336210

  3. 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

  4. Protein Labelling with Versatile Phosphorescent Metal Complexes for Live Cell Luminescence Imaging.

    PubMed

    Connell, Timothy U; James, Janine L; White, Anthony R; Donnelly, Paul S

    2015-09-28

    To take advantage of the luminescent properties of d(6) transition metal complexes to label proteins, versatile bifunctional ligands were prepared. Ligands that contain a 1,2,3-triazole heterocycle were synthesised using Cu(I) catalysed azide-alkyne cycloaddition "click" chemistry and were used to form phosphorescent Ir(III) and Ru(II) complexes. Their emission properties were readily tuned, by changing either the metal ion or the co-ligands. The complexes were tethered to the metalloprotein transferrin using several conjugation strategies. The Ir(III)/Ru(II)-protein conjugates could be visualised in cancer cells using live cell imaging for extended periods without significant photobleaching. These versatile phosphorescent protein-labelling agents could be widely applied to other proteins and biomolecules and are useful alternatives to conventional organic fluorophores for several applications. PMID:26264214

  5. 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

  6. 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

  7. 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.

  8. Neural progenitor cells labeling with microbubble contrast agent for ultrasound imaging in vivo

    PubMed Central

    Cui, Wenjin; Tavri, Sidhartha; Benchimol, Michael J.; Itani, Malak; Olson, Emilia S.; Zhang, Hong; Decyk, Marika; Ramirez, Rosemarie G.; Barback, Christopher V.; Kono, Yuko; Mattrey, Robert F.

    2013-01-01

    Tracking neuroprogenitor cells (NPCs) that are used to target tumors, infarction or inflammation, is paramount for cell-based therapy. We employed ultrasound imaging that can detect a single microbubble because it can distinguish its unique signal from those of surrounding tissues. NPCs efficiently internalized positively charged microbubbles allowing a clinical ultrasound system to detect a single cell at 7 MHz. When injected intravenously, labeled NPCs traversed the lungs to be imaged in the left ventricle and the liver where they accumulated. Internalized microbubbles were not only less sensitive to destruction by ultrasound, but remained visible in vivo for days as compared to minutes when given free. The extended longevity provides ample time to allow cells to reach their intended target. We were also able to transfect NPCs in vitro when microbubbles were preloaded with GFP plasmid only when cells were insonated. Transfection efficiency and cell viability were both greater than 90%. PMID:23578557

  9. Expression profiling. Combinatorial labeling of single cells for gene expression cytometry.

    PubMed

    Fan, H Christina; Fu, Glenn K; Fodor, Stephen P A

    2015-02-01

    We present a technically simple approach for gene expression cytometry combining next-generation sequencing with stochastic barcoding of single cells. A combinatorial library of beads bearing cell- and molecular-barcoding capture probes is used to uniquely label transcripts and reconstruct the digital gene expression profile of thousands of individual cells in a single experiment without the need for robotics or automation. We applied the technology to dissect the human hematopoietic system and to characterize heterogeneous response to in vitro stimulation. High sensitivity is demonstrated by detection of low-abundance transcripts and rare cells. Under current implementation, the technique can analyze a few thousand cells simultaneously and can readily scale to 10,000s or 100,000s of cells. PMID:25657253

  10. 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