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

Optimization and validation of FePro cell labeling method.

PubMed

Janic, Branislava; Rad, Ali M; Jordan, Elaine K; Iskander, A S M; Ali, Md M; Varma, N Ravi S; Frank, Joseph A; Arbab, Ali S

2009-06-11

Current method to magnetically label cells using ferumoxides (Fe)-protamine (Pro) sulfate (FePro) is based on generating FePro complexes in a serum free media that are then incubated overnight with cells for the efficient labeling. However, this labeling technique requires long (>12-16 hours) incubation time and uses relatively high dose of Pro (5-6 microg/ml) that makes large extracellular FePro complexes. These complexes can be difficult to clean with simple cell washes and may create low signal intensity on T2* weighted MRI that is not desirable. The purpose of this study was to revise the current labeling method by using low dose of Pro and adding Fe and Pro directly to the cells before generating any FePro complexes. Human tumor glioma (U251) and human monocytic leukemia cell (THP-1) lines were used as model systems for attached and suspension cell types, respectively and dose dependent (Fe 25 to 100 microg/ml and Pro 0.75 to 3 microg/ml) and time dependent (2 to 48 h) labeling experiments were performed. Labeling efficiency and cell viability of these cells were assessed. Prussian blue staining revealed that more than 95% of cells were labeled. Intracellular iron concentration in U251 cells reached approximately 30-35 pg-iron/cell at 24 h when labeled with 100 microg/ml of Fe and 3 microg/ml of Pro. However, comparable labeling was observed after 4 h across the described FePro concentrations. Similarly, THP-1 cells achieved approximately 10 pg-iron/cell at 48 h when labeled with 100 microg/ml of Fe and 3 microg/ml of Pro. Again, comparable labeling was observed after 4 h for the described FePro concentrations. FePro labeling did not significantly affect cell viability. There was almost no extracellular FePro complexes observed after simple cell washes. To validate and to determine the effectiveness of the revised technique, human T-cells, human hematopoietic stem cells (hHSC), human bone marrow stromal cells (hMSC) and mouse neuronal stem cells (mNSC C17

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

19F-perfluorocarbon-labeled human peripheral blood mononuclear cells can be detected in vivo using clinical MRI parameters in a therapeutic cell setting.

PubMed

Fink, Corby; Gaudet, Jeffrey M; Fox, Matthew S; Bhatt, Shashank; Viswanathan, Sowmya; Smith, Michael; Chin, Joseph; Foster, Paula J; Dekaban, Gregory A

2018-01-12

A 19 Fluorine ( 19 F) perfluorocarbon cell labeling agent, when employed with an appropriate cellular MRI protocol, allows for in vivo cell tracking. 19 F cellular MRI can be used to non-invasively assess the location and persistence of cell-based cancer vaccines and other cell-based therapies. This study was designed to determine the feasibility of labeling and tracking peripheral blood mononuclear cells (PBMC), a heterogeneous cell population. Under GMP-compliant conditions human PBMC were labeled with a 19 F-based MRI cell-labeling agent in a manner safe for autologous re-injection. Greater than 99% of PBMC labeled with the 19 F cell-labeling agent without affecting functionality or affecting viability. The 19 F-labeled PBMC were detected in vivo in a mouse model at the injection site and in a draining lymph node. A clinical cellular MR protocol was optimized for the detection of PBMC injected both at the surface of a porcine shank and at a depth of 1.2 cm, equivalent to depth of a human lymph node, using a dual 1 H/ 19 F dual switchable surface radio frequency coil. This study demonstrates it is feasible to label and track 19 F-labeled PBMC using clinical MRI protocols. Thus, 19 F cellular MRI represents a non-invasive imaging technique suitable to assess the effectiveness of cell-based cancer vaccines.

Controlled viable release of selectively captured label-free cells in microchannels.

PubMed

Gurkan, Umut Atakan; Anand, Tarini; Tas, Huseyin; Elkan, David; Akay, Altug; Keles, Hasan Onur; Demirci, Utkan

2011-12-07

Selective capture of cells from bodily fluids in microchannels has broadly transformed medicine enabling circulating tumor cell isolation, rapid CD4(+) cell counting for HIV monitoring, and diagnosis of infectious diseases. Although cell capture methods have been demonstrated in microfluidic systems, the release of captured cells remains a significant challenge. Viable retrieval of captured label-free cells in microchannels will enable a new era in biological sciences by allowing cultivation and post-processing. The significant challenge in release comes from the fact that the cells adhere strongly to the microchannel surface, especially when immuno-based immobilization methods are used. Even though fluid shear and enzymes have been used to detach captured cells in microchannels, these methods are known to harm cells and affect cellular characteristics. This paper describes a new technology to release the selectively captured label-free cells in microchannels without the use of fluid shear or enzymes. We have successfully released the captured CD4(+) cells (3.6% of the mononuclear blood cells) from blood in microfluidic channels with high specificity (89% ± 8%), viability (94% ± 4%), and release efficiency (59% ± 4%). We have further validated our system by specifically capturing and controllably releasing the CD34(+) stem cells from whole blood, which were quantified to be 19 cells per million blood cells in the blood samples used in this study. Our results also indicated that both CD4(+) and CD34(+) cells released from the microchannels were healthy and amenable for in vitro culture. Manual flow based microfluidic method utilizes inexpensive, easy to fabricate microchannels allowing selective label-free cell capture and release in less than 10 minutes, which can also be used at the point-of-care. The presented technology can be used to isolate and purify a broad spectrum of cells from mixed populations offering widespread applications in applied biological

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.

Autonomous magnetic labelling of functional mesenchymal stem cells for improved traceability and spatial control in cell therapy applications.

PubMed

Harrison, Richard; Markides, Hareklea; Morris, Robert H; Richards, Paula; El Haj, Alicia J; Sottile, Virginie

2017-08-01

Mesenchymal stem cells (MSCs) represent a valuable resource for regenerative medicine treatments for orthopaedic repair and beyond. Following developments in isolation, expansion and differentiation protocols, efforts to promote clinical translation of emerging cellular strategies now seek to improve cell delivery and targeting. This study shows efficient live MSC labelling using silica-coated magnetic particles (MPs), which enables 3D tracking and guidance of stem cells. A procedure developed for the efficient and unassisted particle uptake was shown to support MSC viability and integrity, while surface marker expression and MSC differentiation capability were also maintained. In vitro, MSCs showed a progressive decrease in labelling over increasing culture time, which appeared to be linked to the dilution effect of cell division, rather than to particle release, and did not lead to detectable secondary particle uptake. Labelled MSC populations demonstrated magnetic responsiveness in vitro through directed migration in culture and, when seeded onto a scaffold, supporting MP-based approaches to cell targeting. The potential of these silica-coated MPs for MRI cell tracking of MSC populations was validated in 2D and in a cartilage repair model following cell delivery. These results highlight silica-coated magnetic particles as a simple, safe and effective resource to enhance MSC targeting for therapeutic applications and improve patient outcomes. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.

Carbon "Quantum" Dots for Fluorescence Labeling of Cells.

PubMed

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

2015-09-02

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

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

Whole body tracking of superparamagnetic iron oxide nanoparticle-labelled cells – a rheumatoid arthritis mouse model

PubMed Central

2013-01-01

Introduction The application of mesenchymal stem cells (MSCs) in treating rheumatoid arthritis (RA) has been made possible by the immunosuppressive and differentiation abilities of these cells. A non-invasive means of assessing cell integration and bio-distribution is fundamental in evaluating the risks and success of this therapy, thereby enabling clinical translation. This paper defines the use of superparamagnetic iron oxide nanoparticles (SPIONs) in conjunction with magnetic resonance imaging (MRI) to image and track MSCs in vivo within a murine model of RA. Methods Murine MSCs (mMSCs) were isolated, expanded and labelled with SiMAG, a commercially available particle. In vitro MRI visibility thresholds were investigated by labelling mMSCs with SiMAG with concentrations ranging from 0 to 10 μg/ml and resuspending varying cell doses (103 to 5 × 105 cells) in 2 mg/ml collagen prior to MR-imaging. Similarly, in vivo detection thresholds were identified by implanting 3 × 105 mMSCs labelled with 0 to 10 μg/ml SiMAG within the synovial cavity of a mouse and MR-imaging. Upon RA induction, 300,000 mMSCs labelled with SiMAG (10 μg/ml) were implanted via intra-articular injection and joint swelling monitored as an indication of RA development over seven days. Furthermore, the effect of SiMAG on cell viability, proliferation and differentiation was investigated. Results A minimum particle concentration of 1 μg/ml (300,000 cells) and cell dose of 100,000 cells (5 and 10 μg/ml) were identified as the in vitro MRI detection threshold. Cell viability, proliferation and differentiation capabilities were not affected, with labelled populations undergoing successful differentiation down osteogenic and adipogenic lineages. A significant decrease (P < 0.01) in joint swelling was measured in groups containing SiMAG-labelled and unlabelled mMSCs implying that the presence of SPIONs does not affect the immunomodulating properties of the cells. In vivo MRI

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

Quantification of Superparamagnetic Iron Oxide (SPIO)-labeled Cells Using MRI

PubMed Central

Rad, Ali M; Arbab, Ali S; Iskander, ASM; Jiang, Quan; Soltanian-Zadeh, Hamid

2015-01-01

Purpose To show the feasibility of using magnetic resonance imaging (MRI) to quantify superparamagnetic iron oxide (SPIO)-labeled cells. Materials and Methods Lymphocytes and 9L rat gliosarcoma cells were labeled with Ferumoxides-Protamine Sulfate complex (FE-PRO). Cells were labeled efficiently (more than 95%) and iron concentration inside each cell was measured by spectrophotometry (4.77-30.21 picograms). Phantom tubes containing different number of labeled or unlabeled cells as well as different concentrations of FE-PRO were made. In addition, labeled and unlabeled cells were injected into fresh and fixed rat brains. Results Cellular viability and proliferation of labeled and unlabeled cells were shown to be similar. T2-weighted images were acquired using 7 T and 3 T MRI systems and R2 maps of the tubes containing cells, free FE-PRO, and brains were made. There was a strong linear correlation between R2 values and labeled cell numbers but the regression lines were different for the lymphocytes and gliosarcoma cells. Similarly, there was strong correlation between R2 values and free iron. However, free iron had higher R2 values than the labeled cells for the same concentration of iron. Conclusion Our data indicated that in vivo quantification of labeled cells can be done by careful consideration of different factors and specific control groups. PMID:17623892

Isolation of mouse pancreatic alpha, beta, duct and acinar populations with cell surface markers.

PubMed

Dorrell, Craig; Grompe, Maria T; Pan, Fong Cheng; Zhong, Yongping; Canaday, Pamela S; Shultz, Leonard D; Greiner, Dale L; Wright, Chris V; Streeter, Philip R; Grompe, Markus

2011-06-06

Tools permitting the isolation of live pancreatic cell subsets for culture and/or molecular analysis are limited. To address this, we developed a collection of monoclonal antibodies with selective surface labeling of endocrine and exocrine pancreatic cell types. Cell type labeling specificity and cell surface reactivity were validated on mouse pancreatic sections and by gene expression analysis of cells isolated using FACS. Five antibodies which marked populations of particular interest were used to isolate and study viable populations of purified pancreatic ducts, acinar cells, and subsets of acinar cells from whole pancreatic tissue or of alpha or beta cells from isolated mouse islets. Gene expression analysis showed the presence of known endocrine markers in alpha and beta cell populations and revealed that TTR and DPPIV are primarily expressed in alpha cells whereas DGKB and GPM6A have a beta cell specific expression profile. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Cell-specific Labeling Enzymes for Analysis of Cell–Cell Communication in Continuous Co-culture*

PubMed Central

Tape, Christopher J.; Norrie, Ida C.; Worboys, Jonathan D.; Lim, Lindsay; Lauffenburger, Douglas A.; Jørgensen, Claus

2014-01-01

We report the orthologous screening, engineering, and optimization of amino acid conversion enzymes for cell-specific proteomic labeling. Intracellular endoplasmic-reticulum-anchored Mycobacterium tuberculosis diaminopimelate decarboxylase (DDCM.tub-KDEL) confers cell-specific meso-2,6-diaminopimelate-dependent proliferation to multiple eukaryotic cell types. Optimized lysine racemase (LyrM37-KDEL) supports D-lysine specific proliferation and efficient cell-specific isotopic labeling. When ectopically expressed in discrete cell types, these enzymes confer 90% cell-specific isotopic labeling efficiency after 10 days of co-culture. Moreover, DDCM.tub-KDEL and LyrM37-KDEL facilitate equally high cell-specific labeling fidelity without daily media exchange. Consequently, the reported novel enzyme pairing can be used to study cell-specific signaling in uninterrupted, continuous co-cultures. Demonstrating the importance of increased labeling stability for addressing novel biological questions, we compare the cell-specific phosphoproteome of fibroblasts in direct co-culture with epithelial tumor cells in both interrupted (daily media exchange) and continuous (no media exchange) co-cultures. This analysis identified multiple cell-specific phosphorylation sites specifically regulated in the continuous co-culture. Given their applicability to multiple cell types, continuous co-culture labeling fidelity, and suitability for long-term cell–cell phospho-signaling experiments, we propose DDCM.tub-KDEL and LyrM37-KDEL as excellent enzymes for cell-specific labeling with amino acid precursors. PMID:24820872

Identification of epithelial label-retaining cells at the transition between the anal canal and the rectum in mice

PubMed Central

Runck, Laura A; Kramer, Megan; Ciraolo, Georgianne; Lewis, Alfor G

2010-01-01

In certain regions of the body, transition zones exist where stratified squamous epithelia directly abut against other types of epithelia. Certain transition zones are especially prone to tumorigenesis an example being the anorectal junction, although the reason for this is not known. One possibility is that the abrupt transition of the simple columnar epithelium of the colon to the stratified squamous epithelium of the proximal portion of the anal canal may contain a unique stem cell niche. We investigated whether the anorectal region contained cells with stem cell properties relative to the adjacent epithelium. We utilized a tetracycline-regulatable histone H2B-GFP transgenic mice model, previously used to identify hair follicle stem cells, to fluorescently label slow-cycling anal epithelial cells (e.g., prospective stem cells) in combination with a panel of putative stem cell markers. We identified a population of long-term GFP label-retaining cells concentrated at the junction between the anal canal and the rectum. These cells are BrdU-retaining cells and expressed the stem cell marker CD34. Moreover, tracking the fate of the anal label-retaining cells in vivo revealed that the slow-cycling cells only gave rise to progeny of the anal epithelium. In conclusion, we identified a unique population of cells at the anorectal junction which can be separated from the other basal anal epithelial cells based upon the expression of the stem cell marker CD34 and integrin α6, and thus represent a putative anal stem cell population. PMID:20647777

Stem Cell Monitoring with a Direct or Indirect Labeling Method.

PubMed

Kim, Min Hwan; Lee, Yong Jin; Kang, Joo Hyun

2016-12-01

The molecular imaging techniques allow monitoring of the transplanted cells in the same individuals over time, from early localization to the survival, migration, and differentiation. Generally, there are two methods of stem cell labeling: direct and indirect labeling methods. The direct labeling method introduces a labeling agent into the cell, which is stably incorporated or attached to the cells prior to transplantation. Direct labeling of cells with radionuclides is a simple method with relatively fewer adverse events related to genetic responses. However, it can only allow short-term distribution of transplanted cells because of the decreasing imaging signal with radiodecay, according to the physical half-lives, or the signal becomes more diffuse with cell division and dispersion. The indirect labeling method is based on the expression of a reporter gene transduced into the cell before transplantation, which is then visualized upon the injection of an appropriate probe or substrate. In this review, various imaging strategies to monitor the survival and behavior change of transplanted stem cells are covered. Taking these new approaches together, the direct and indirect labeling methods may provide new insights on the roles of in vivo stem cell monitoring, from bench to bedside.

Label-free single-cell separation and imaging of cancer cells using an integrated microfluidic system.

PubMed

Antfolk, Maria; Kim, Soo Hyeon; Koizumi, Saori; Fujii, Teruo; Laurell, Thomas

2017-04-20

The incidence of cancer is increasing worldwide and metastatic disease, through the spread of circulating tumor cells (CTCs), is responsible for the majority of the cancer deaths. Accurate monitoring of CTC levels in blood provides clinical information supporting therapeutic decision making, and improved methods for CTC enumeration are asked for. Microfluidics has been extensively used for this purpose but most methods require several post-separation processing steps including concentration of the sample before analysis. This induces a high risk of sample loss of the collected rare cells. Here, an integrated system is presented that efficiently eliminates this risk by integrating label-free separation with single cell arraying of the target cell population, enabling direct on-chip tumor cell identification and enumeration. Prostate cancer cells (DU145) spiked into a sample with whole blood concentration of the peripheral blood mononuclear cell (PBMC) fraction were efficiently separated and trapped at a recovery of 76.2 ± 5.9% of the cancer cells and a minute contamination of 0.12 ± 0.04% PBMCs while simultaneously enabling a 20x volumetric concentration. This constitutes a first step towards a fully integrated system for rapid label-free separation and on-chip phenotypic characterization of circulating tumor cells from peripheral venous blood in clinical practice.

Cell-selective metabolic labeling of biomolecules with bioorthogonal functionalities.

PubMed

Xie, Ran; Hong, Senlian; Chen, Xing

2013-10-01

Metabolic labeling of biomolecules with bioorthogonal functionalities enables visualization, enrichment, and analysis of the biomolecules of interest in their physiological environments. This versatile strategy has found utility in probing various classes of biomolecules in a broad range of biological processes. On the other hand, metabolic labeling is nonselective with respect to cell type, which imposes limitations for studies performed in complex biological systems. Herein, we review the recent methodological developments aiming to endow metabolic labeling strategies with cell-type selectivity. The cell-selective metabolic labeling strategies have emerged from protein and glycan labeling. We envision that these strategies can be readily extended to labeling of other classes of biomolecules. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

PubMed

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

2015-01-01

Magnetic resonance imaging (MRI) provides an effective approach to track labeled pluripotent stem cell (PSC)-derived neural progenitor cells (NPCs) for neurological disorder treatments after cell labeling with a contrast agent, such as an iron oxide derivative. Cryopreservation of pre-labeled neural cells, especially in three-dimensional (3D) structure, can provide a uniform cell population and preserve the stem cell niche for the subsequent applications. In this study, the effects of cryopreservation on PSC-derived multicellular NPC aggregates labeled with micron-sized particles of iron oxide (MPIO) were investigated. These NPC aggregates were labeled prior to cryopreservation because labeling thawed cells can be limited by inefficient intracellular uptake, variations in labeling efficiency, and increased culture time before use, minimizing their translation to clinical settings. The results indicated that intracellular MPIO incorporation was retained after cryopreservation (70-80% labeling efficiency), and MPIO labeling had little adverse effects on cell recovery, proliferation, cytotoxicity and neural lineage commitment post-cryopreservation. MRI analysis showed comparable detectability for the MPIO-labeled cells before and after cryopreservation indicated by T2 and T2* relaxation rates. Cryopreserving MPIO-labeled 3D multicellular NPC aggregates can be applied in in vivo cell tracking studies and lead to more rapid translation from preservation to clinical implementation. © 2015 American Institute of Chemical Engineers.

Label-free single-cell separation and imaging of cancer cells using an integrated microfluidic system

PubMed Central

Antfolk, Maria; Kim, Soo Hyeon; Koizumi, Saori; Fujii, Teruo; Laurell, Thomas

2017-01-01

The incidence of cancer is increasing worldwide and metastatic disease, through the spread of circulating tumor cells (CTCs), is responsible for the majority of the cancer deaths. Accurate monitoring of CTC levels in blood provides clinical information supporting therapeutic decision making, and improved methods for CTC enumeration are asked for. Microfluidics has been extensively used for this purpose but most methods require several post-separation processing steps including concentration of the sample before analysis. This induces a high risk of sample loss of the collected rare cells. Here, an integrated system is presented that efficiently eliminates this risk by integrating label-free separation with single cell arraying of the target cell population, enabling direct on-chip tumor cell identification and enumeration. Prostate cancer cells (DU145) spiked into a sample with whole blood concentration of the peripheral blood mononuclear cell (PBMC) fraction were efficiently separated and trapped at a recovery of 76.2 ± 5.9% of the cancer cells and a minute contamination of 0.12 ± 0.04% PBMCs while simultaneously enabling a 20x volumetric concentration. This constitutes a first step towards a fully integrated system for rapid label-free separation and on-chip phenotypic characterization of circulating tumor cells from peripheral venous blood in clinical practice. PMID:28425472

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

Cell-to-cell variation and specialization in sugar metabolism in clonal bacterial populations

PubMed Central

Schreiber, Frank; Dal Co, Alma; Kiviet, Daniel J.; Littmann, Sten

2017-01-01

While we have good understanding of bacterial metabolism at the population level, we know little about the metabolic behavior of individual cells: do single cells in clonal populations sometimes specialize on different metabolic pathways? Such metabolic specialization could be driven by stochastic gene expression and could provide individual cells with growth benefits of specialization. We measured the degree of phenotypic specialization in two parallel metabolic pathways, the assimilation of glucose and arabinose. We grew Escherichia coli in chemostats, and used isotope-labeled sugars in combination with nanometer-scale secondary ion mass spectrometry and mathematical modeling to quantify sugar assimilation at the single-cell level. We found large variation in metabolic activities between single cells, both in absolute assimilation and in the degree to which individual cells specialize in the assimilation of different sugars. Analysis of transcriptional reporters indicated that this variation was at least partially based on cell-to-cell variation in gene expression. Metabolic differences between cells in clonal populations could potentially reduce metabolic incompatibilities between different pathways, and increase the rate at which parallel reactions can be performed. PMID:29253903

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

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

Deep Learning in Label-free Cell Classification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia

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

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

Deep Learning in Label-free Cell Classification

DOE PAGES

Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; ...

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

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.

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

NASA Astrophysics Data System (ADS)

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

2016-03-01

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

Magnetic-Activated Cell Sorting for the Fast and Efficient Separation of Human and Rodent Schwann Cells from Mixed Cell Populations.

PubMed

Ravelo, Kristine M; Andersen, Natalia D; Monje, Paula V

2018-01-01

To date, magnetic-activated cell sorting (MACS) remains a powerful method to isolate distinct cell populations based on differential cell surface labeling. Optimized direct and indirect MACS protocols for cell immunolabeling are presented here as methods to divest Schwann cell (SC) cultures of contaminating cells (specifically, fibroblast cells) and isolate SC populations at different stages of differentiation. This chapter describes (1) the preparation of single-cell suspensions from established human and rat SC cultures, (2) the design and application of cell selection strategies using SC-specific (p75 NGFR , O4, and O1) and fibroblast-specific (Thy-1) markers, and (3) the characterization of both the pre- and post-sorting cell populations. A simple protocol for the growth of hybridoma cell cultures as a source of monoclonal antibodies for cell surface immunolabeling of SCs and fibroblasts is provided as a cost-effective alternative for commercially available products. These steps allow for the timely and efficient recovery of purified SC populations without compromising the viability and biological activity of the cells.

Affinity Versus Label-Free Isolation of Circulating Tumor Cells: Who Wins?

PubMed

Murlidhar, Vasudha; Rivera-Báez, Lianette; Nagrath, Sunitha

2016-09-01

The study of circulating tumor cells (CTCs) has been made possible by many technological advances in their isolation. Their isolation has seen many fronts, but each technology brings forth a new set of challenges to overcome. Microfluidics has been a key player in the capture of CTCs and their downstream analysis, with the aim of shedding light into their clinical application in cancer and metastasis. Researchers have taken diverging paths to isolate such cells from blood, ranging from affinity-based isolation targeting surface antigens expressed on CTCs, to label-free isolation taking advantage of the size differences between CTCs and other blood cells. For both major groups, many microfluidic technologies have reported high sensitivity and specificity for capturing CTCs. However, the question remains as to the superiority among these two isolation techniques, specifically to identify different CTC populations. This review highlights the key aspects of affinity and label-free microfluidic CTC technologies, and discusses which of these two would be the highest benefactor for the study of CTCs. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Chronology of Islet Differentiation Revealed By Temporal Cell Labeling

PubMed Central

Miyatsuka, Takeshi; Li, Zhongmei; German, Michael S.

2009-01-01

OBJECTIVE Neurogenin 3 plays a pivotal role in pancreatic endocrine differentiation. Whereas mouse models expressing reporters such as eGFP or LacZ under the control of the Neurog3 gene enable us to label cells in the pancreatic endocrine lineage, the long half-life of most reporter proteins makes it difficult to distinguish cells actively expressing neurogenin 3 from differentiated cells that have stopped transcribing the gene. RESEARCH DESIGN AND METHODS In order to separate the transient neurogenin 3 –expressing endocrine progenitor cells from the differentiating endocrine cells, we developed a mouse model (Ngn3-Timer) in which DsRed-E5, a fluorescent protein that shifts its emission spectrum from green to red over time, was expressed transgenically from the NEUROG3 locus. RESULTS In the Ngn3-Timer embryos, green-dominant cells could be readily detected by microscopy or flow cytometry and distinguished from green/red double-positive cells. When fluorescent cells were sorted into three different populations by a fluorescence-activated cell sorter, placed in culture, and then reanalyzed by flow cytometry, green-dominant cells converted to green/red double-positive cells within 6 h. The sorted cell populations were then used to determine the temporal patterns of expression for 145 transcriptional regulators in the developing pancreas. CONCLUSIONS The precise temporal resolution of this model defines the narrow window of neurogenin 3 expression in islet progenitor cells and permits sequential analyses of sorted cells as well as the testing of gene regulatory models for the differentiation of pancreatic islet cells. PMID:19478145

Enhanced labeling density and whole-cell 3D dSTORM imaging by repetitive labeling of target proteins.

PubMed

Venkataramani, Varun; Kardorff, Markus; Herrmannsdörfer, Frank; Wieneke, Ralph; Klein, Alina; Tampé, Robert; Heilemann, Mike; Kuner, Thomas

2018-04-03

With continuing advances in the resolving power of super-resolution microscopy, the inefficient labeling of proteins with suitable fluorophores becomes a limiting factor. For example, the low labeling density achieved with antibodies or small molecule tags limits attempts to reveal local protein nano-architecture of cellular compartments. On the other hand, high laser intensities cause photobleaching within and nearby an imaged region, thereby further reducing labeling density and impairing multi-plane whole-cell 3D super-resolution imaging. Here, we show that both labeling density and photobleaching can be addressed by repetitive application of trisNTA-fluorophore conjugates reversibly binding to a histidine-tagged protein by a novel approach called single-epitope repetitive imaging (SERI). For single-plane super-resolution microscopy, we demonstrate that, after multiple rounds of labeling and imaging, the signal density is increased. Using the same approach of repetitive imaging, washing and re-labeling, we demonstrate whole-cell 3D super-resolution imaging compensated for photobleaching above or below the imaging plane. This proof-of-principle study demonstrates that repetitive labeling of histidine-tagged proteins provides a versatile solution to break the 'labeling barrier' and to bypass photobleaching in multi-plane, whole-cell 3D experiments.

Selective in vivo metabolic cell-labeling-mediated cancer targeting

PubMed Central

Wang, Hua; Wang, Ruibo; Cai, Kaimin; He, Hua; Liu, Yang; Yen, Jonathan; Wang, Zhiyu; Xu, Ming; Sun, Yiwen; Zhou, Xin; Yin, Qian; Tang, Li; Dobrucki, Iwona T; Dobrucki, Lawrence W; Chaney, Eric J; Boppart, Stephen A; Fan, Timothy M; Lezmi, Stéphane; Chen, Xuesi; Yin, Lichen; Cheng, Jianjun

2017-01-01

Distinguishing cancer cells from normal cells through surface receptors is vital for cancer diagnosis and targeted therapy. Metabolic glycoengineering of unnatural sugars provides a powerful tool to manually introduce chemical receptors onto the cell surface; however, cancer-selective labeling still remains a great challenge. Herein we report the design of sugars that can selectively label cancer cells both in vitro and in vivo. Specifically, we inhibit the cell-labeling activity of tetraacetyl-N-azidoacetylmannosamine (Ac4ManAz) by converting its anomeric acetyl group to a caged ether bond that can be selectively cleaved by cancer-overexpressed enzymes and thus enables the overexpression of azido groups on the surface of cancer cells. Histone deacetylase and cathepsin L-responsive acetylated azidomannosamine, one such enzymatically activatable Ac4ManAz analog developed, mediated cancer-selective labeling in vivo, which enhanced tumor accumulation of a dibenzocyclooctyne–doxorubicin conjugate via click chemistry and enabled targeted therapy against LS174T colon cancer, MDA-MB-231 triple-negative breast cancer and 4T1 metastatic breast cancer in mice. PMID:28192414

Adeno Associated Viral-mediated intraosseus labeling of bone marrow derived cells for CNS tracking

PubMed Central

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

2016-01-01

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 intraosseus 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

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

PubMed

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

2015-03-03

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

Ferritin conjugates as specific magnetic labels. Implications for cell separation.

PubMed Central

Odette, L L; McCloskey, M A; Young, S H

1984-01-01

Concanavalin A coupled to the naturally occurring iron storage protein ferritin is used to label rat erythrocytes and increase the cells' magnetic susceptibility. Labeled cells are introduced into a chamber containing spherical iron particles and the chamber is placed in a uniform 5.2 kG (gauss) magnetic field. The trajectory of cells in the inhomogeneous magnetic field around the iron particles and the polar distributions of cells bound to the iron particles compare well with the theoretical predictions for high gradient magnetic systems. On the basis of these findings we suggest that ferritin conjugated ligands can be used for selective magnetic separation of labeled cells. Images FIGURE 2 PMID:6743752

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.

Stem Cell-Like Differentiation Potentials of Endometrial Side Population Cells as Revealed by a Newly Developed In Vivo Endometrial Stem Cell Assay

PubMed Central

Miyazaki, Kaoru; Maruyama, Tetsuo; Masuda, Hirotaka; Yamasaki, Akiko; Uchida, Sayaka; Oda, Hideyuki; Uchida, Hiroshi; Yoshimura, Yasunori

2012-01-01

Background Endometrial stem/progenitor cells contribute to the cyclical regeneration of human endometrium throughout a woman's reproductive life. Although the candidate cell populations have been extensively studied, no consensus exists regarding which endometrial population represents the stem/progenitor cell fraction in terms of in vivo stem cell activity. We have previously reported that human endometrial side population cells (ESP), but not endometrial main population cells (EMP), exhibit stem cell-like properties, including in vivo reconstitution of endometrium-like tissues when xenotransplanted into immunodeficient mice. The reconstitution efficiency, however, was low presumably because ESP cells alone could not provide a sufficient microenvironment (niche) to support their stem cell activity. The objective of this study was to establish a novel in vivo endometrial stem cell assay employing cell tracking and tissue reconstitution systems and to examine the stem cell properties of ESP through use of this assay. Methodology/Principal Findings ESP and EMP cells isolated from whole endometrial cells were infected with lentivirus to express tandem Tomato (TdTom), a red fluorescent protein. They were mixed with unlabeled whole endometrial cells and then transplanted under the kidney capsule of ovariectomized immunodeficient mice. These mice were treated with estradiol and progesterone for eight weeks and nephrectomized. All of the grafts reconstituted endometrium-like tissues under the kidney capsules. Immunofluorescence revealed that TdTom-positive cells were significantly more abundant in the glandular, stromal, and endothelial cells of the reconstituted endometrium in mice transplanted with TdTom-labeled ESP cells than those with TdTom-labeled EMP cells. Conclusions/Significance We have established a novel in vivo endometrial stem cell assay in which multi-potential differentiation can be identified through cell tracking during in vivo endometrial tissue

Label Structured Cell Proliferation Models

DTIC Science & Technology

2010-06-16

and (, + ) are the cell proliferation and death rates , respectively, relative to the moving label coordinate system + . Daughter...proliferation and death rates relative to this new coordinate system. While not common in the biological sciences, it is altogether common in the physical

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

PubMed

Hayashi, Takahiro; Hamachi, Itaru

2012-09-18

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

Large-scale detection of antigen-specific T cells using peptide-MHC-I multimers labeled with DNA barcodes.

PubMed

Bentzen, Amalie Kai; Marquard, Andrea Marion; Lyngaa, Rikke; Saini, Sunil Kumar; Ramskov, Sofie; Donia, Marco; Such, Lina; Furness, Andrew J S; McGranahan, Nicholas; Rosenthal, Rachel; Straten, Per Thor; Szallasi, Zoltan; Svane, Inge Marie; Swanton, Charles; Quezada, Sergio A; Jakobsen, Søren Nyboe; Eklund, Aron Charles; Hadrup, Sine Reker

2016-10-01

Identification of the peptides recognized by individual T cells is important for understanding and treating immune-related diseases. Current cytometry-based approaches are limited to the simultaneous screening of 10-100 distinct T-cell specificities in one sample. Here we use peptide-major histocompatibility complex (MHC) multimers labeled with individual DNA barcodes to screen >1,000 peptide specificities in a single sample, and detect low-frequency CD8 T cells specific for virus- or cancer-restricted antigens. When analyzing T-cell recognition of shared melanoma antigens before and after adoptive cell therapy in melanoma patients, we observe a greater number of melanoma-specific T-cell populations compared with cytometry-based approaches. Furthermore, we detect neoepitope-specific T cells in tumor-infiltrating lymphocytes and peripheral blood from patients with non-small cell lung cancer. Barcode-labeled pMHC multimers enable the combination of functional T-cell analysis with large-scale epitope recognition profiling for the characterization of T-cell recognition in various diseases, including in small clinical samples.

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.

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

PubMed Central

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

2014-01-01

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

Direct fluorescent-dye labeling of α-tubulin in mammalian cells for live cell and superresolution imaging

PubMed Central

Schvartz, Tomer; Aloush, Noa; Goliand, Inna; Segal, Inbar; Nachmias, Dikla; Arbely, Eyal; Elia, Natalie

2017-01-01

Genetic code expansion and bioorthogonal labeling provide for the first time a way for direct, site-specific labeling of proteins with fluorescent-dyes in live cells. Although the small size and superb photophysical parameters of fluorescent-dyes offer unique advantages for high-resolution microscopy, this approach has yet to be embraced as a tool in live cell imaging. Here we evaluated the feasibility of this approach by applying it for α-tubulin labeling. After a series of calibrations, we site-specifically labeled α-tubulin with silicon rhodamine (SiR) in live mammalian cells in an efficient and robust manner. SiR-labeled tubulin successfully incorporated into endogenous microtubules at high density, enabling video recording of microtubule dynamics in interphase and mitotic cells. Applying this labeling approach to structured illumination microscopy resulted in an increase in resolution, highlighting the advantages in using a smaller, brighter tag. Therefore, using our optimized assay, genetic code expansion provides an attractive tool for labeling proteins with a minimal, bright tag in quantitative high-resolution imaging. PMID:28835375

Selective dye-labeling of newly synthesized proteins in bacterial cells.

PubMed

Beatty, Kimberly E; Xie, Fang; Wang, Qian; Tirrell, David A

2005-10-19

We describe fluorescence labeling of newly synthesized proteins in Escherichia coli cells by means of Cu(I)-catalyzed cycloaddition between alkynyl amino acid side chains and the fluorogenic dye 3-azido-7-hydroxycoumarin. The method involves co-translational labeling of proteins by the non-natural amino acids homopropargylglycine (Hpg) or ethynylphenylalanine (Eth) followed by treatment with the dye. As a demonstration, the model protein barstar was expressed and treated overnight with Cu(I) and 3-azido-7-hydroxycoumarin. Examination of treated cells by confocal microscopy revealed that strong fluorescence enhancement was observed only for alkynyl-barstar treated with Cu(I) and the reactive dye. The cellular fluorescence was punctate, and gel electrophoresis confirmed that labeled barstar was localized in inclusion bodies. Other proteins showed little fluorescence. Examination of treated cells by fluorimetry demonstrated that cultures supplemented with Eth or Hpg showed an 8- to 14-fold enhancement in fluorescence intensity after labeling. Addition of a protein synthesis inhibitor reduced the emission intensity to levels slightly above background, confirming selective labeling of newly synthesized proteins in the bacterial cell.

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

PubMed

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

2016-05-01

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

Single-Cell Growth Rates in Photoautotrophic Populations Measured by Stable Isotope Probing and Resonance Raman Microspectrometry

PubMed Central

Taylor, Gordon T.; Suter, Elizabeth A.; Li, Zhuo Q.; Chow, Stephanie; Stinton, Dallyce; Zaliznyak, Tatiana; Beaupré, Steven R.

2017-01-01

A new method to measure growth rates of individual photoautotrophic cells by combining stable isotope probing (SIP) and single-cell resonance Raman microspectrometry is introduced. This report explores optimal experimental design and the theoretical underpinnings for quantitative responses of Raman spectra to cellular isotopic composition. Resonance Raman spectra of isogenic cultures of the cyanobacterium, Synechococcus sp., grown in 13C-bicarbonate revealed linear covariance between wavenumber (cm−1) shifts in dominant carotenoid Raman peaks and a broad range of cellular 13C fractional isotopic abundance. Single-cell growth rates were calculated from spectra-derived isotopic content and empirical relationships. Growth rates among any 25 cells in a sample varied considerably; mean coefficient of variation, CV, was 29 ± 3% (σ/x¯), of which only ~2% was propagated analytical error. Instantaneous population growth rates measured independently by in vivo fluorescence also varied daily (CV ≈ 53%) and were statistically indistinguishable from single-cell growth rates at all but the lowest levels of cell labeling. SCRR censuses of mixtures prepared from Synechococcus sp. and T. pseudonana (a diatom) populations with varying 13C-content and growth rates closely approximated predicted spectral responses and fractional labeling of cells added to the sample. This approach enables direct microspectrometric interrogation of isotopically- and phylogenetically-labeled cells and detects as little as 3% changes in cellular fractional labeling. This is the first description of a non-destructive technique to measure single-cell photoautotrophic growth rates based on Raman spectroscopy and well-constrained assumptions, while requiring few ancillary measurements. PMID:28824580

Pulse labeling of RNA of mammalian cells.

PubMed

Rovera, G; Berman, S; Baserga, R

1970-04-01

When cells from a hypotetraploid strain of Ehrlich ascites tumor are exposed to uridine-(3)H either in vivo or in vitro, the amount of radioactivity incorporated into RNA reaches a maximum within ten minutes, after which any further incorporation stops. (3)H-uridine triphosphate disappears from the acid soluble pool within 30 minutes and the findings indicate that the RNA of these cells can be pulse labeled without the use of any antibiotic or the need of a "chase." The stability of the pulse labeled RNA in the presence of pentobarbital (an inhibitor of RNA synthesis) indicates the virtual absence of RNA breakdown. However, actinomycin D, at a dosage of 250 mug/mouse in vivo and 10 mug/ml in vitro produces breakdown of labeled RNA, thus confirming earlier observations that the drug is not a suitable tool for RNA kinetics determinations. The pulse-labeled RNA leaves the nucleus slowly and some radioactive RNA is still present in the nuclear fraction after 24 hours. Radioactivity begins to appear in cytoplasmic ribosomal RNA after 20 minutes and continues to increase up to six hours.

Siloxane nanoprobes for labeling and dual modality imaging of neural stem cells

PubMed Central

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

2015-01-01

Cell therapy represents a promising therapeutic for a myriad of medical conditions, including cancer, traumatic brain injury, and cardiovascular disease among others. A thorough understanding of the efficacy and cellular dynamics of these therapies necessitates the ability to non-invasively track cells in vivo. Magnetic resonance imaging (MRI) provides a platform to track cells as a non-invasive modality with superior resolution and soft tissue contrast. We recently reported a new nanoprobe platform for cell labeling and imaging using fluorophore doped siloxane core nanoemulsions as dual modality (1H MRI/Fluorescence), dual-functional (oximetry/detection) nanoprobes. Here, we successfully demonstrate the labeling, dual-modality imaging, and oximetry of neural progenitor/stem cells (NPSCs) in vitro using this platform. Labeling at a concentration of 10 μl/104 cells with a 40%v/v polydimethylsiloxane core nanoemulsion, doped with rhodamine, had minimal effect on viability, no effect on migration, proliferation and differentiation of NPSCs and allowed for unambiguous visualization of labeled NPSCs by 1H MR and fluorescence and local pO2 reporting by labeled NPSCs. This new approach for cell labeling with a positive contrast 1H MR probe has the potential to improve mechanistic knowledge of current therapies, and guide the design of future cell therapies due to its clinical translatability. PMID:26597417

Mesenchymal Stem Cell Preparation and Transfection-free Ferumoxytol Labeling for MRI Cell Tracking.

PubMed

Liu, Li; Ho, Chien

2017-11-15

Mesenchymal stem cells (MSCs) are multipotent cells and are the most widely studied cell type for stem cell therapies. In vivo cell tracking of MSCs labeled with an FDA-approved superparamagnetic iron-oxide (SPIO) particle by magnetic resonance imaging (MRI) provides essential information, e.g., MSC engraftment, survival, and fate, thus improving cell therapy accuracy. However, current methodology for labeling MSCs with Ferumoxytol (Feraheme ® ), the only FDA-approved SPIO particle, needs transfection agents. This unit describes a new "bio-mimicry" protocol to prepare more native MSCs by using more "in vivo environment" of MSCs, so that the phagocytic activity of cultured MSCs is restored and expanded MSCs can be labeled with Ferumoxytol, without the need for transfection agents and/or electroporation. Moreover, MSCs re-size to a more native size, reducing from 32.0 to 19.5 μm. The MSCs prepared from this protocol retain more native properties and would be useful for biomedical applications and MSC-tracking studies by MRI. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

In silico lineage tracing through single cell transcriptomics identifies a neural stem cell population in planarians.

PubMed

Molinaro, Alyssa M; Pearson, Bret J

2016-04-27

The planarian Schmidtea mediterranea is a master regenerator with a large adult stem cell compartment. The lack of transgenic labeling techniques in this animal has hindered the study of lineage progression and has made understanding the mechanisms of tissue regeneration a challenge. However, recent advances in single-cell transcriptomics and analysis methods allow for the discovery of novel cell lineages as differentiation progresses from stem cell to terminally differentiated cell. Here we apply pseudotime analysis and single-cell transcriptomics to identify adult stem cells belonging to specific cellular lineages and identify novel candidate genes for future in vivo lineage studies. We purify 168 single stem and progeny cells from the planarian head, which were subjected to single-cell RNA sequencing (scRNAseq). Pseudotime analysis with Waterfall and gene set enrichment analysis predicts a molecularly distinct neoblast sub-population with neural character (νNeoblasts) as well as a novel alternative lineage. Using the predicted νNeoblast markers, we demonstrate that a novel proliferative stem cell population exists adjacent to the brain. scRNAseq coupled with in silico lineage analysis offers a new approach for studying lineage progression in planarians. The lineages identified here are extracted from a highly heterogeneous dataset with minimal prior knowledge of planarian lineages, demonstrating that lineage purification by transgenic labeling is not a prerequisite for this approach. The identification of the νNeoblast lineage demonstrates the usefulness of the planarian system for computationally predicting cellular lineages in an adult context coupled with in vivo verification.

Magneto-optical labeling of fetal neural stem cells for in vivo MRI tracking.

PubMed

Flexman, J A; Minoshima, S; Kim, Y; Cross, D J

2006-01-01

Neural stem cell therapy for neurological pathologies, such as Alzheimer's and Parkinson's disease, may delay the onset of symptoms, replace damaged neurons and/or support the survival of endogenous cells. Magnetic resonance imaging (MRI) can be used to track magnetically labeled cells in vivo to observe migration. Prior to transplantation, labeled cells must be characterized to show that they retain their intrinsic properties, such as cell proliferation into neurospheres in a supplemented environment. In vivo images must also be correlated to sensitive, histological markers. In this study, we show that fetus-derived neural stem cells can be co-labeled with superparamagnetic iron oxide and PKH26, a fluorescent dye. Labeled cells retain the ability to proliferate into neurospheres in culture, but labeling prevents neurospheres from merging in a non-adherent culture environment. After labeled NSCs were transplantation into the rat brain, their location and subsequent migration along the corpus callosum was detected using MRI. This study demonstrates an imaging paradigm with which to develop an in vivo assay for quantitatively evaluating fetal neural stem cell migration.

Direct fluorescent-dye labeling of α-tubulin in mammalian cells for live cell and superresolution imaging.

PubMed

Schvartz, Tomer; Aloush, Noa; Goliand, Inna; Segal, Inbar; Nachmias, Dikla; Arbely, Eyal; Elia, Natalie

2017-10-15

Genetic code expansion and bioorthogonal labeling provide for the first time a way for direct, site-specific labeling of proteins with fluorescent-dyes in live cells. Although the small size and superb photophysical parameters of fluorescent-dyes offer unique advantages for high-resolution microscopy, this approach has yet to be embraced as a tool in live cell imaging. Here we evaluated the feasibility of this approach by applying it for α-tubulin labeling. After a series of calibrations, we site-specifically labeled α-tubulin with silicon rhodamine (SiR) in live mammalian cells in an efficient and robust manner. SiR-labeled tubulin successfully incorporated into endogenous microtubules at high density, enabling video recording of microtubule dynamics in interphase and mitotic cells. Applying this labeling approach to structured illumination microscopy resulted in an increase in resolution, highlighting the advantages in using a smaller, brighter tag. Therefore, using our optimized assay, genetic code expansion provides an attractive tool for labeling proteins with a minimal, bright tag in quantitative high-resolution imaging. © 2017 Schvartz et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Human Adipose-Derived Stem Cells Labeled with Plasmonic Gold Nanostars for Cellular Tracking and Photothermal Cancer Cell Ablation.

PubMed

Shammas, Ronnie L; Fales, Andrew M; Crawford, Bridget M; Wisdom, Amy J; Devi, Gayathri R; Brown, David A; Vo-Dinh, Tuan; Hollenbeck, Scott T

2017-04-01

Gold nanostars are unique nanoplatforms that can be imaged in real time and transform light energy into heat to ablate cells. Adipose-derived stem cells migrate toward tumor niches in response to chemokines. The ability of adipose-derived stem cells to migrate and integrate into tumors makes them ideal vehicles for the targeted delivery of cancer nanotherapeutics. To test the labeling efficiency of gold nanostars, undifferentiated adipose-derived stem cells were incubated with gold nanostars and a commercially available nanoparticle (Qtracker), then imaged using two-photon photoluminescence microscopy. The effects of gold nanostars on cell phenotype, proliferation, and viability were assessed with flow cytometry, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide metabolic assay, and trypan blue, respectively. Trilineage differentiation of gold nanostar-labeled adipose-derived stem cells was induced with the appropriate media. Photothermolysis was performed on adipose-derived stem cells cultured alone or in co-culture with SKBR3 cancer cells. Efficient uptake of gold nanostars occurred in adipose-derived stem cells, with persistence of the luminescent signal over 4 days. Labeling efficiency and signal quality were greater than with Qtracker. Gold nanostars did not affect cell phenotype, viability, or proliferation, and exhibited stronger luminescence than Qtracker throughout differentiation. Zones of complete ablation surrounding the gold nanostar-labeled adipose-derived stem cells were observed following photothermolysis in both monoculture and co-culture models. Gold nanostars effectively label adipose-derived stem cells without altering cell phenotype. Once labeled, photoactivation of gold nanostar-labeled adipose-derived stem cells ablates neighboring cancer cells, demonstrating the potential of adipose-derived stem cells as a vehicle for the delivery of site-specific cancer therapy.

Labeling tetracysteine-tagged proteins with biarsenical dyes for live cell imaging.

PubMed

Gaietta, Guido M; Deerinck, Thomas J; Ellisman, Mark H

2011-01-01

Correlation of real-time or time-lapse light microscopy (LM) with electron microscopy (EM) of cells can be performed with biarsenical dyes. These dyes fluorescently label tetracysteine-tagged proteins so that they can be imaged with LM and, upon fluorescent photoconversion of 3,3'-diaminobenzidine tetrahydrochloride (DAB), with EM as well. In the following protocol, cells expressing tetracysteine-tagged proteins are labeled for 1 h with biarsenical dyes. The volumes indicated are for a single 30-mm culture dish containing 2 mL of labeling medium. Scale the suggested volumes up or down depending upon the size of the culture dish used in the labeling. The same procedure can be adapted for longer labeling times by lowering the amount of dye used to 50-100 nM; however, the amount of the competing dithiol EDT is maintained at 10-20 μM. Longer labeling times often produce higher signal-to-noise ratios and cause less trauma to the treated cells prior to imaging.

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.

Viability and proliferation potential of adipose-derived stem cells following labeling with a positron-emitting radiotracer.

PubMed

Elhami, Esmat; Goertzen, Andrew L; Xiang, Bo; Deng, Jixian; Stillwell, Chris; Mzengeza, Shadreck; Arora, Rakesh C; Freed, Darren; Tian, Ganghong

2011-07-01

Adipose-derived stem cells (ASCs) have promising potential in regenerative medicine and cell therapy. Our objective is to examine the biological function of the labeled stem cells following labeling with a readily available positron emission tomography (PET) tracer, (18)F-fluoro-2-deoxy-D: -glucose (FDG). In this work we characterize labeling efficiency through assessment of FDG uptake and retention by the ASCs and the effect of FDG on cell viability, proliferation, transdifferentiation, and cell function in vitro using rat ASCs. Samples of 10(5) ASCs (from visceral fat tissue) were labeled with concentrations of FDG (1-55 Bq/cell) in 0.75 ml culture medium. Label uptake and retention, as a function of labeling time, FDG concentration, and efflux period were measured to determine optimum cell labeling conditions. Cell viability, proliferation, DNA structure damage, cell differentiation, and other cell functions were examined. Non-labeled ASC samples were used as a control for all experimental groups. Labeled ASCs were injected via tail vein in several healthy rats and initial cell biodistribution was assessed. Our results showed that FDG uptake and retention by the stem cells did not depend on FDG concentration but on labeling and efflux periods and glucose content of the labeling and efflux media. Cell viability, transdifferentiation, and cell function were not greatly affected. DNA damage due to FDG radioactivity was acute, but reversible; cells managed to repair the damage and continue with cell cycles. Over all, FDG (up to 25 Bq/cell) did not impose severe cytotoxicity in rat ASCs. Initial biodistribution of the FDG-labeled ASCs was 80% + retention in the lungs. In the delayed whole-body images (2-3 h postinjection) there was some activity distribution resembling typical FDG uptake patterns. For in vivo cell tracking studies with PET tracers, the parameter of interest is the amount of radiotracer that is present in the cells being labeled and consequent

Label-free and live cell imaging by interferometric scattering microscopy.

PubMed

Park, Jin-Sung; Lee, Il-Buem; Moon, Hyeon-Min; Joo, Jong-Hyeon; Kim, Kyoung-Hoon; Hong, Seok-Cheol; Cho, Minhaeng

2018-03-14

Despite recent remarkable advances in microscopic techniques, it still remains very challenging to directly observe the complex structure of cytoplasmic organelles in live cells without a fluorescent label. Here we report label-free and live-cell imaging of mammalian cell, Escherischia coli , and yeast, using interferometric scattering microscopy, which reveals the underlying structures of a variety of cytoplasmic organelles as well as the underside structure of the cells. The contact areas of the cells attached onto a glass substrate, e.g. , focal adhesions and filopodia, are clearly discernible. We also found a variety of fringe-like features in the cytoplasmic area, which may reflect the folded structures of cytoplasmic organelles. We thus anticipate that the label-free interferometric scattering microscopy can be used as a powerful tool to shed interferometric light on in vivo structures and dynamics of various intracellular phenomena.

Magnetic field design for selecting and aligning immunomagnetic labeled cells.

PubMed

Tibbe, Arjan G J; de Grooth, Bart G; Greve, Jan; Dolan, Gerald J; Rao, Chandra; Terstappen, Leon W M M

2002-03-01

Recently we introduced the CellTracks cell analysis system, in which samples are prepared based on a combination of immunomagnetic selection, separation, and alignment of cells along ferromagnetic lines. Here we describe the underlying magnetic principles and considerations made in the magnetic field design to achieve the best possible cell selection and alignment of magnetically labeled cells. Materials and Methods Computer simulations, in combination with experimental data, were used to optimize the design of the magnets and Ni lines to obtain the optimal magnetic configuration. A homogeneous cell distribution on the upper surface of the sample chamber was obtained with a magnet where the pole faces were tilted towards each other. The spatial distribution of magnetically aligned objects in between the Ni lines was dependent on the ratio of the diameter of the aligned object and the line spacing, which was tested with magnetically and fluorescently labeled 6 microm polystyrene beads. The best result was obtained when the line spacing was equal to or smaller than the diameter of the aligned object. The magnetic gradient of the designed permanent magnet extracts magnetically labeled cells from any cell suspension to a desired plane, providing a homogeneous cell distribution. In addition, it magnetizes ferro-magnetic Ni lines in this plane whose additional local gradient adds to the gradient of the permanent magnet. The resultant gradient aligns the magnetically labeled cells first brought to this plane. This combination makes it possible, in a single step, to extract and align cells on a surface from any cell suspension. Copyright 2002 Wiley-Liss, Inc.

Developments in label-free microfluidic methods for single-cell analysis and sorting.

PubMed

Carey, Thomas R; Cotner, Kristen L; Li, Brian; Sohn, Lydia L

2018-04-24

Advancements in microfluidic technologies have led to the development of many new tools for both the characterization and sorting of single cells without the need for exogenous labels. Label-free microfluidics reduce the preparation time, reagents needed, and cost of conventional methods based on fluorescent or magnetic labels. Furthermore, these devices enable analysis of cell properties such as mechanical phenotype and dielectric parameters that cannot be characterized with traditional labels. Some of the most promising technologies for current and future development toward label-free, single-cell analysis and sorting include electronic sensors such as Coulter counters and electrical impedance cytometry; deformation analysis using optical traps and deformation cytometry; hydrodynamic sorting such as deterministic lateral displacement, inertial focusing, and microvortex trapping; and acoustic sorting using traveling or standing surface acoustic waves. These label-free microfluidic methods have been used to screen, sort, and analyze cells for a wide range of biomedical and clinical applications, including cell cycle monitoring, rapid complete blood counts, cancer diagnosis, metastatic progression monitoring, HIV and parasite detection, circulating tumor cell isolation, and point-of-care diagnostics. Because of the versatility of label-free methods for characterization and sorting, the low-cost nature of microfluidics, and the rapid prototyping capabilities of modern microfabrication, we expect this class of technology to continue to be an area of high research interest going forward. New developments in this field will contribute to the ongoing paradigm shift in cell analysis and sorting technologies toward label-free microfluidic devices, enabling new capabilities in biomedical research tools as well as clinical diagnostics. This article is categorized under: Diagnostic Tools > Biosensing Diagnostic Tools > Diagnostic Nanodevices. © 2018 Wiley Periodicals, Inc.

Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells.

PubMed

Peng, Tao; Hang, Howard C

2016-11-02

Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse-electron-demand Diels-Alder cycloaddition reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.

Combining Raman spectroscopy and digital holographic microscopy for label-free classification of human immune cells (Conference Presentation)

NASA Astrophysics Data System (ADS)

McReynolds, Naomi; Cooke, Fiona G. M.; Chen, Mingzhou; Powis, Simon J.; Dholakia, Kishan

2017-02-01

Moving towards label-free techniques for cell identification is essential for many clinical and research applications. Raman spectroscopy and digital holographic microscopy (DHM) are both label-free, non-destructive optical techniques capable of providing complimentary information. We demonstrate a multi-modal system which may simultaneously take Raman spectra and DHM images to provide both a molecular and a morphological description of our sample. In this study we use Raman spectroscopy and DHM to discriminate between three immune cell populations CD4+ T cells, B cells, and monocytes, which together comprise key functional immune cell subsets in immune responses to invading pathogens. Various parameters that may be used to describe the phase images are also examined such as pixel value histograms or texture analysis. Using our system it is possible to consider each technique individually or in combination. Principal component analysis is used on the data set to discriminate between cell types and leave-one-out cross-validation is used to estimate the efficiency of our method. Raman spectroscopy provides specific chemical information but requires relatively long acquisition times, combining this with a faster modality such as DHM could help achieve faster throughput rates. The combination of these two complimentary optical techniques provides a wealth of information for cell characterisation which is a step towards achieving label free technology for the identification of human immune cells.

Label-free resistive-pulse cytometry.

PubMed

Chapman, M R; Sohn, L L

2011-01-01

Numerous methods have recently been developed to characterize cells for size, shape, and specific cell-surface markers. Most of these methods rely upon exogenous labeling of the cells and are better suited for large cell populations (>10,000). Here, we review a label-free method of characterizing and screening cells based on the Coulter-counter technique of particle sizing: an individual cell transiting a microchannel (or "pore") causes a downward pulse in the measured DC current across that "pore". Pulse magnitude corresponds to the cell size, pulse width to the transit time needed for the cell to pass through the pore, and pulse shape to how the cell traverses across the pore (i.e., rolling or tumbling). When the pore is functionalized with an antibody that is specific to a surface-epitope of interest, label-free screening of a specific marker is possible, as transient binding between the two results in longer time duration than when the pore is unfunctionalized or functionalized with a nonspecific antibody. While this method cannot currently compete with traditional technology in terms of throughput, there are a number of applications for which this technology is better suited than current commercial cytometry systems. Applications include the rapid and nondestructive analysis of small cell populations (<100), which is not possible with current technology, and a platform for providing true point-of-care clinical diagnostics, due to the simplicity of the device, low manufacturing costs, and ease of use. Copyright © 2011 Elsevier Inc. All rights reserved.

Minocycline Causes Widespread Cell Death and Increases Microglial Labeling in the Neonatal Mouse Brain

PubMed Central

Strahan, J. Alex; Walker, William H.; Montgomery, Taylor R.; Forger, Nancy G.

2016-01-01

Minocycline, an antibiotic of the tetracycline family, inhibits microglia in many paradigms, and is among the most commonly used tools for examining the role of microglia in physiological processes. Microglia may play an active role in triggering developmental neuronal cell death, although findings have been contradictory. To determine whether microglia influence developmental cell death, we treated perinatal mice with minocycline (45 mg/kg) and quantified effects on dying cells and microglial labeling using immunohistochemistry for activated caspase-3 (AC3) and ionized calcium-binding adapter molecule 1 (Iba1), respectively. Contrary to our expectations, minocycline treatment from embryonic day 18 to postnatal day (P)1 caused a >10-fold increase in cell death 8 h after the last injection in all brain regions examined, including the primary sensory cortex (S1), septum, hippocampus and hypothalamus. Iba1 labeling was also increased in most regions. Similar effects, although of smaller magnitude, were seen when treatment was delayed to P3-P5. Minocycline treatment from P3-P5 also decreased overall cell number in the septum at weaning, suggesting lasting effects of the neonatal exposure. When administered at lower doses (4.5 or 22.5 mg/kg), or at the same dose one week later (P10-P12), minocycline no longer increased microglial markers or cell death. Taken together, the most commonly used microglial “inhibitor” increases cell death and Iba1 labeling in the neonatal mouse brain. Minocycline is used clinically in infant and pediatric populations; caution is warrented when using minocycline in developing animals, or extrapolating the effects of this drug across ages. PMID:27706925

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

Stripe-patterned thermo-responsive cell culture dish for cell separation without cell labeling.

PubMed

Kumashiro, Yoshikazu; Ishihara, Jun; Umemoto, Terumasa; Itoga, Kazuyoshi; Kobayashi, Jun; Shimizu, Tatsuya; Yamato, Masayuki; Okano, Teruo

2015-02-11

A stripe-patterned thermo-responsive surface is prepared to enable cell separation without labeling. The thermo-responsive surface containing a 3 μm striped pattern exhibits various cell adhesion and detachment properties. A mixture of three cell types is separated on the patterned surface based on their distinct cell-adhesion properties, and the composition of the cells is analyzed by flow cytometry. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Labeling proteins inside living cells using external fluorophores for microscopy.

PubMed

Teng, Kai Wen; Ishitsuka, Yuji; Ren, Pin; Youn, Yeoan; Deng, Xiang; Ge, Pinghua; Lee, Sang Hak; Belmont, Andrew S; Selvin, Paul R

2016-12-09

Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial enzyme which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG's to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20-30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes.

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

Modelling the balance between quiescence and cell death in normal and tumour cell populations.

PubMed

Spinelli, Lorenzo; Torricelli, Alessandro; Ubezio, Paolo; Basse, Britta

2006-08-01

When considering either human adult tissues (in vivo) or cell cultures (in vitro), cell number is regulated by the relationship between quiescent cells, proliferating cells, cell death and other controls of cell cycle duration. By formulating a mathematical description we see that even small alterations of this relationship may cause a non-growing population to start growing with doubling times characteristic of human tumours. Our model consists of two age structured partial differential equations for the proliferating and quiescent cell compartments. Model parameters are death rates from and transition rates between these compartments. The partial differential equations can be solved for the steady-age distributions, giving the distribution of the cells through the cell cycle, dependent on specific model parameter values. Appropriate formulas can then be derived for various population characteristic quantities such as labelling index, proliferation fraction, doubling time and potential doubling time of the cell population. Such characteristic quantities can be estimated experimentally, although with decreasing precision from in vitro, to in vivo experimental systems and to the clinic. The model can be used to investigate the effects of a single alteration of either quiescence or cell death control on the growth of the whole population and the non-trivial dependence of the doubling time and other observable quantities on particular underlying cell cycle scenarios of death and quiescence. The model indicates that tumour evolution in vivo is a sequence of steady-states, each characterised by particular death and quiescence rate functions. We suggest that a key passage of carcinogenesis is a loss of the communication between quiescence, death and cell cycle machineries, causing a defect in their precise, cell cycle dependent relationship.

In Situ Live-Cell Nucleus Fluorescence Labeling with Bioinspired Fluorescent Probes.

PubMed

Ding, Pan; Wang, Houyu; Song, Bin; Ji, Xiaoyuan; Su, Yuanyuan; He, Yao

2017-08-01

Fluorescent imaging techniques for visualization of nuclear structure and function in live cells are fundamentally important for exploring major cellular events. The ideal cellular labeling method is capable of realizing label-free, in situ, real-time, and long-term nucleus labeling in live cells, which can fully obtain the nucleus-relative information and effectively alleviate negative effects of alien probes on cellular metabolism. However, current established fluorescent probes-based strategies (e.g., fluorescent proteins-, organic dyes-, fluorescent organic/inorganic nanoparticles-based imaging techniques) are unable to simultaneously realize label-free, in situ, long-term, and real-time nucleus labeling, resulting in inevitable difficulties in fully visualizing nuclear structure and function in live cells. To this end, we present a type of bioinspired fluorescent probes, which are highly efficacious for in situ and label-free tracking of nucleus in long-term and real-time manners. Typically, the bioinspired polydopamine (PDA) nanoparticles, served as fluorescent probes, can be readily synthesized in situ within live cell nucleus without any further modifications under physiological conditions (37 °C, pH ∼7.4). Compared with other conventional nuclear dyes (e.g., propidium iodide (PI), Hoechst), superior spectroscopic properties (e.g., quantum yield of ∼35.8% and high photostability) and low cytotoxicity of PDA-based probes enable long-term (e.g., 3 h) fluorescence tracking of nucleus. We also demonstrate the generality of this type of bioinspired fluorescent probes in different cell lines and complex biological samples.

Label-free detection of HIV-1 infected cells via integration of optical tweezers and photoluminescence spectroscopy

NASA Astrophysics Data System (ADS)

Lugongolo, Masixole Yvonne; Ombinda-Lemboumba, Saturnin; Noto, Luyanda Lunga; Maaza, Malik; Mthunzi-Kufa, Patience

2018-02-01

The human immunodeficiency virus-1 (HIV-1) is currently detected using conventional qualitative and quantitative tests to determine the presence or absence of HIV in blood samples. However, the approach of these tests detects the presence of either viral antibodies or viral RNA that require labelling which may be costly, sophisticated and time consuming. A label-free approach of detecting the presence of HIV is therefore desirable. Of note optical tweezers can be coupled with other technologies including spectroscopy, which also investigates light-matter interactions. For example, coupling of optical tweezers with luminescence spectroscopy techniques has emerged as a powerful tool in biology for micro-manipulation, detection and analysis of individual cells. Integration of optical techniques has enabled studying biological particles in a label-free manner, whilst detecting functional groups and other essential molecules within mixed populations of cells. In the current study, an optical trapping system coupled to luminescence spectroscopy was utilised to detect the presence of HIV infection in TZM-bl cells in vitro. This was performed by infecting TZM-bl cells with the ZM53 HIV-1 pseudovirus, and incubating them for 48 hours prior analysis. The differences between infected and uninfected cells were thereafter displayed as shown by the spectrographs obtained. Combination of these two techniques has a potential in the field of infectious disease diagnostics.

Labeling single cell for in-vivo study of cell fate mapping and lineage tracing

NASA Astrophysics Data System (ADS)

He, Sicong; Xu, Jin; Wu, Yi; Tian, Ye; Sun, Qiqi; Wen, Zilong; Qu, Jianan Y.

2018-02-01

Cell fate mapping and lineage tracing are significant ways to understanding the developmental origins of biological tissues. It requires labeling individual cells and tracing the development of their progeny. We develop an infrared laser-evoked gene operator heat-shock microscope system to achieve single-cell labeling in zebrafish. With a fluorescent thermometry technique, we measure the temperature increase in zebrafish tissues induced by infrared laser and identify the optimal heat shock conditions for single-cell gene induction in different types of zebrafish cells. We use this technique to study the fate mapping of T lymphocytes and discover the distinct waves of lymphopoiesis during the zebrafish development.

Single cell systems biology by super-resolution imaging and combinatorial labeling

PubMed Central

Lubeck, Eric; Cai, Long

2012-01-01

Fluorescence microscopy is a powerful quantitative tool for exploring regulatory networks in single cells. However, the number of molecular species that can be measured simultaneously is limited by the spectral separability of fluorophores. Here we demonstrate a simple but general strategy to drastically increase the capacity for multiplex detection of molecules in single cells by using optical super-resolution microscopy (SRM) and combinatorial labeling. As a proof of principle, we labeled mRNAs with unique combinations of fluorophores using Fluorescence in situ Hybridization (FISH), and resolved the sequences and combinations of fluorophores with SRM. We measured the mRNA levels of 32 genes simultaneously in single S. cerevisiae cells. These experiments demonstrate that combinatorial labeling and super-resolution imaging of single cells provides a natural approach to bring systems biology into single cells. PMID:22660740

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

NASA Astrophysics Data System (ADS)

Chandra, Subhash

2004-06-01

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

Prolonged Dye Release from Mesoporous Silica-Based Imaging Probes Facilitates Long-Term Optical Tracking of Cell Populations In Vivo.

PubMed

Rosenholm, Jessica M; Gulin-Sarfraz, Tina; Mamaeva, Veronika; Niemi, Rasmus; Özliseli, Ezgi; Desai, Diti; Antfolk, Daniel; von Haartman, Eva; Lindberg, Desiré; Prabhakar, Neeraj; Näreoja, Tuomas; Sahlgren, Cecilia

2016-03-23

Nanomedicine is gaining ground worldwide in therapy and diagnostics. Novel nanoscopic imaging probes serve as imaging tools for studying dynamic biological processes in vitro and in vivo. To allow detectability in the physiological environment, the nanostructure-based probes need to be either inherently detectable by biomedical imaging techniques, or serve as carriers for existing imaging agents. In this study, the potential of mesoporous silica nanoparticles carrying commercially available fluorochromes as self-regenerating cell labels for long-term cellular tracking is investigated. The particle surface is organically modified for enhanced cellular uptake, the fluorescence intensity of labeled cells is followed over time both in vitro and in vivo. The particles are not exocytosed and particles which escaped cells due to cell injury or death are degraded and no labeling of nontargeted cell populations are observed. The labeling efficiency is significantly improved as compared to that of quantum dots of similar emission wavelength. Labeled human breast cancer cells are xenotransplanted in nude mice, and the fluorescent cells can be detected in vivo for a period of 1 month. Moreover, ex vivo analysis reveals fluorescently labeled metastatic colonies in lymph node and rib, highlighting the capability of the developed probes for tracking of metastasis. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fluorescently-labeled RNA packaging into HIV-1 particles: Direct examination of infectivity across central nervous system cell types.

PubMed

Xu, Ruqiang; El-Hage, Nazira; Dever, Seth M

2015-11-01

HIV penetrates the central nervous system (CNS), and although it is clear that microglia and to a lesser extent astrocytes are infected, whether certain other cell types such as neurons are infected remains unclear. Here, we confirmed the finding that RNAs of both cellular and viral origins are present in native HIV-1 particles and exploited this phenomenon to directly examine HIV-1 infectivity of CNS cell types. Using in vitro transcribed mRNAs that were labeled with a fluorescent dye, we showed that these fluorescent mRNAs were packaged into HIV-1 particles by directly examining infected cells using fluorescence microscopy. Cells in culture infected with these labeled virions showed the fluorescent signals of mRNA labels by a distinct pattern of punctate, focal signals within the cells which was used to demonstrate that the CXCR4-tropic NL4-3 strain was able to enter microglia and to a lesser extent astrocytes, but not neurons. The strategy used in the present study may represent a novel approach of simplicity, robustness and reliability for versatile applications in HIV studies, such as the determination of infectivity across a broad range of cell types and within sub-populations of an individual cell type by direct visualization of viral entry into cells. Copyright © 2015 Elsevier B.V. All rights reserved.

The Protein Corona around Nanoparticles Facilitates Stem Cell Labeling for Clinical MR Imaging.

PubMed

Nejadnik, Hossein; Taghavi-Garmestani, Seyed-Meghdad; Madsen, Steven J; Li, Kai; Zanganeh, Saeid; Yang, Phillip; Mahmoudi, Morteza; Daldrup-Link, Heike E

2018-03-01

Purpose To evaluate if the formation of a protein corona around ferumoxytol nanoparticles can facilitate stem cell labeling for in vivo tracking with magnetic resonance (MR) imaging. Materials and Methods Ferumoxytol was incubated in media containing human serum (group 1), fetal bovine serum (group 2), StemPro medium (group 3), protamine (group 4), and protamine plus heparin (group 5). Formation of a protein corona was characterized by means of dynamic light scattering, ζ potential, and liquid chromatography-mass spectrometry. Iron uptake was evaluated with 3,3'-diaminobenzidine-Prussian blue staining, lysosomal staining, and inductively coupled plasma spectrometry. To evaluate the effect of a protein corona on stem cell labeling, human mesenchymal stem cells (hMSCs) were labeled with the above formulations, implanted into pig knee specimens, and investigated with T2-weighted fast spin-echo and multiecho spin-echo sequences on a 3.0-T MR imaging unit. Data in different groups were compared by using a Kruskal-Wallis test. Results Compared with bare nanoparticles, all experimental groups showed significantly increased negative ζ values (from -37 to less than -10; P = .008). Nanoparticles in groups 1-3 showed an increased size because of the formation of a protein corona. hMSCs labeled with group 1-5 media showed significantly shortened T2 relaxation times compared with unlabeled control cells (P = .0012). hMSCs labeled with group 3 and 5 media had the highest iron uptake after cells labeled with group 1 medium. After implantation into pig knees, hMSCs labeled with group 1 medium showed significantly shorter T2 relaxation times than hMSCs labeled with group 2-5 media (P = .0022). Conclusion The protein corona around ferumoxytol nanoparticles can facilitate stem cell labeling for clinical cell tracking with MR imaging. © RSNA, 2017 Online supplemental material is available for this article.

Minocycline causes widespread cell death and increases microglial labeling in the neonatal mouse brain.

PubMed

Strahan, J Alex; Walker, William H; Montgomery, Taylor R; Forger, Nancy G

2017-06-01

Minocycline, an antibiotic of the tetracycline family, inhibits microglia in many paradigms and is among the most commonly used tools for examining the role of microglia in physiological processes. Microglia may play an active role in triggering developmental neuronal cell death, although findings have been contradictory. To determine whether microglia influence developmental cell death, we treated perinatal mice with minocycline (45 mg/kg) and quantified effects on dying cells and microglial labeling using immunohistochemistry for activated caspase-3 (AC3) and ionized calcium-binding adapter molecule 1 (Iba1), respectively. Contrary to our expectations, minocycline treatment from embryonic day 18 to postnatal day (P)1 caused a > tenfold increase in cell death 8 h after the last injection in all brain regions examined, including the primary sensory cortex, septum, hippocampus and hypothalamus. Iba1 labeling was also increased in most regions. Similar effects, although of smaller magnitude, were seen when treatment was delayed to P3-P5. Minocycline treatment from P3 to P5 also decreased overall cell number in the septum at weaning, suggesting lasting effects of the neonatal exposure. When administered at lower doses (4.5 or 22.5 mg/kg), or at the same dose 1 week later (P10-P12), minocycline no longer increased microglial markers or cell death. Taken together, the most commonly used microglial "inhibitor" increases cell death and Iba1 labeling in the neonatal mouse brain. Minocycline is used clinically in infant and pediatric populations; caution is warrented when using minocycline in developing animals, or extrapolating the effects of this drug across ages. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 753-766, 2017. © 2016 Wiley Periodicals, Inc.

Stable isotope labeling by essential nutrients in cell culture for preparation of labeled coenzyme A and its thioesters.

PubMed

Basu, Sankha S; Mesaros, Clementina; Gelhaus, Stacy L; Blair, Ian A

2011-02-15

Stable isotope dilution mass spectrometry (MS) represents the gold standard for quantification of endogenously formed cellular metabolites. Although coenzyme A (CoA) and acyl-CoA thioester derivatives are central players in numerous metabolic pathways, the lack of a commercially available isotopically labeled CoA limits the development of rigorous MS-based methods. In this study, we adapted stable isotope labeling by amino acids in cell culture (SILAC) methodology to biosynthetically generate stable isotope labeled CoA and thioester analogues for use as internal standards in liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) assays. This was accomplished by incubating murine hepatocytes (Hepa 1c1c7) in media in which pantothenate (a precursor of CoA) was replaced with [(13)C(3)(15)N(1)]-pantothenate. Efficient incorporation into various CoA species was optimized to >99% [(13)C(3)(15)N(1)]-pantothenate after three passages of the murine cells in culture. Charcoal-dextran-stripped fetal bovine serum (FBS) was found to be more efficient for serum supplementation than dialyzed or undialyzed FBS, due to lower contaminating unlabeled pantothenate content. Stable isotope labeled CoA species were extracted and utilized as internal standards for CoA thioester analysis in cell culture models. This methodology of stable isotope labeling by essential nutrients in cell culture (SILEC) can serve as a paradigm for using vitamins and other essential nutrients to generate stable isotope standards that cannot be readily synthesized.

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

PubMed

Terada, Takaho; Yokoyama, Shigeyuki

2015-01-01

This chapter describes the cell-free protein synthesis method, using an Escherichia coli cell extract. This is a cost-effective method for milligram-scale protein production and is particularly useful for the production of mammalian proteins, protein complexes, and membrane proteins that are difficult to synthesize by recombinant expression methods, using E. coli and eukaryotic cells. By adjusting the conditions of the cell-free method, zinc-binding proteins, disulfide-bonded proteins, ligand-bound proteins, etc., may also be produced. Stable isotope labeling of proteins can be accomplished by the cell-free method, simply by using stable isotope-labeled amino acid(s) in the cell-free reaction. Moreover, the cell-free protein synthesis method facilitates the avoidance of stable isotope scrambling and dilution over the recombinant expression methods and is therefore advantageous for amino acid-selective stable isotope labeling. Site-specific stable isotope labeling is also possible with a tRNA molecule specific to the UAG codon. By the cell-free protein synthesis method, coupled transcription-translation is performed from a plasmid vector or a PCR-amplified DNA fragment encoding the protein. A milligram quantity of protein can be produced with a milliliter-scale reaction solution in the dialysis mode. More than a thousand solution structures have been determined by NMR spectroscopy for uniformly labeled samples of human and mouse functional domain proteins, produced by the cell-free method. Here, we describe the practical aspects of mammalian protein production by the cell-free method for NMR spectroscopy. © 2015 Elsevier Inc. All rights reserved.

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.

A genetically encoded and gate for cell-targeted metabolic labeling of proteins.

PubMed

Mahdavi, Alborz; Segall-Shapiro, Thomas H; Kou, Songzi; Jindal, Granton A; Hoff, Kevin G; Liu, Shirley; Chitsaz, Mohsen; Ismagilov, Rustem F; Silberg, Jonathan J; Tirrell, David A

2013-02-27

We describe a genetic AND gate for cell-targeted metabolic labeling and proteomic analysis in complex cellular systems. The centerpiece of the AND gate is a bisected methionyl-tRNA synthetase (MetRS) that charges the Met surrogate azidonorleucine (Anl) to tRNA(Met). Cellular protein labeling occurs only upon activation of two different promoters that drive expression of the N- and C-terminal fragments of the bisected MetRS. Anl-labeled proteins can be tagged with fluorescent dyes or affinity reagents via either copper-catalyzed or strain-promoted azide-alkyne cycloaddition. Protein labeling is apparent within 5 min after addition of Anl to bacterial cells in which the AND gate has been activated. This method allows spatial and temporal control of proteomic labeling and identification of proteins made in specific cellular subpopulations. The approach is demonstrated by selective labeling of proteins in bacterial cells immobilized in the center of a laminar-flow microfluidic channel, where they are exposed to overlapping, opposed gradients of inducers of the N- and C-terminal MetRS fragments. The observed labeling profile is predicted accurately from the strengths of the individual input signals.

A Genetically Encoded AND Gate for Cell-Targeted Metabolic Labeling of Proteins

PubMed Central

Mahdavi, Alborz; Segall-Shapiro, Thomas H.; Kou, Songzi; Jindal, Granton A.; Hoff, Kevin G.; Liu, Shirley; Chitsaz, Mohsen; Ismagilov, Rustem F.; Silberg, Jonathan J.; Tirrell, David A.

2013-01-01

We describe a genetic AND gate for cell-targeted metabolic labeling and proteomic analysis in complex cellular systems. The centerpiece of the AND gate is a bisected methionyl-tRNA synthetase (MetRS) that charges the Met surrogate azidonorleucine (Anl) to tRNAMet. Cellular protein labeling occurs only upon activation of two different promoters that drive expression of the N- and C-terminal fragments of the bisected MetRS. Anl-labeled proteins can be tagged with fluorescent dyes or affinity reagents via either copper-catalyzed or strain-promoted azide-alkyne cycloaddition. Protein labeling is apparent within five minutes after addition of Anl to bacterial cells in which the AND gate has been activated. This method allows spatial and temporal control of proteomic labeling and identification of proteins made in specific cellular subpopulations. The approach is demonstrated by selective labeling of proteins in bacterial cells immobilized in the center of a laminar-flow microfluidic channel, where they are exposed to overlapping, opposed gradients of inducers of the N- and C-terminal MetRS fragments. The observed labeling profile is predicted accurately from the strengths of the individual input signals. PMID:23406315

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.

"Peer workers/health counselors": a new label for a labeled population. A work-in-progress which may not progress.

PubMed

Einstein, Stan

2012-04-01

The relatively new labels, "peer workers," "peer health counselors," etc., are explored in terms of a selected number of their implications--few, if any, of which have been or are positive for this targeted population.

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

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

PubMed

Gutmann, E; Mares, V; Stichová, J

1976-03-05

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

In situ label-free quantification of human pluripotent stem cells with electrochemical potential.

PubMed

Yea, Cheol-Heon; Jeong, Ho-Chang; Moon, Sung-Hwan; Lee, Mi-Ok; Kim, Kyeong-Jun; Choi, Jeong-Woo; Cha, Hyuk-Jin

2016-01-01

Conventional methods for quantification of undifferentiated pluripotent stem cells such as fluorescence-activated cell sorting and real-time PCR analysis have technical limitations in terms of their sensitivity and recyclability. Herein, we designed a real-time in situ label-free monitoring system on the basis of a specific electrochemical signature of human pluripotent stem cells in vitro. The intensity of the signal of hPSCs highly corresponded to the cell number and remained consistent in a mixed population with differentiated cells. The electrical charge used for monitoring did not markedly affect the proliferation rate or molecular characteristics of differentiated human aortic smooth muscle cells. After YM155 treatment to ablate undifferentiated hPSCs, their specific signal was significantly reduced. This suggests that detection of the specific electrochemical signature of hPSCs would be a valid approach to monitor potential contamination of undifferentiated hPSCs, which can assess the risk of teratoma formation efficiently and economically. Copyright © 2015 Elsevier Ltd. All rights reserved.

Technetium-99m labeled red blood cells in the evaluation of hemangiosarcoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Joseph, U.A.; Jhingran, S.G.

Imaging with Tc-99m labeled red blood cells (RBC) is increasingly being used in the detection of acute gastro-intestinal bleeding, especially in patients with intermittent bleeding. A patient is presented in whom the labeled RBC scan was helpful in the incidental discovery of a previously unsuspected probable angiosarcoma of the right femur and adjacent soft tissues of the right hip due to the blood pool or blush effect of the labeled cells. The labeled RBC scan also identified extravasation due to active gastrointestinal bleeding from a previously unknown angiosarcoma of the ascending colon. Thus, the Tc-99m labeled RBC scan was usefulmore » in simultaneously detecting extravasation and blood pool effect at two remote tumor sites in the same patient.« less

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

PubMed

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

2014-01-01

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

Fluorine-19 Labeling of Stromal Vascular Fraction Cells for Clinical Imaging Applications

PubMed Central

Rose, Laura C.; Kadayakkara, Deepak K.; Wang, Guan; Bar-Shir, Amnon; Helfer, Brooke M.; O’Hanlon, Charles F.; Kraitchman, Dara L.; Rodriguez, Ricardo L.

2015-01-01

Stromal vascular fraction (SVF) cells are used clinically for various therapeutic targets. The location and persistence of engrafted SVF cells are important parameters for determining treatment failure versus success. We used the GID SVF-1 platform and a clinical protocol to harvest and label SVF cells with the fluorinated (19F) agent CS-1000 as part of a first-in-human phase I trial (clinicaltrials.gov identifier NCT02035085) to track SVF cells with magnetic resonance imaging during treatment of radiation-induced fibrosis in breast cancer patients. Flow cytometry revealed that SVF cells consisted of 25.0% ± 15.8% CD45+, 24.6% ± 12.5% CD34+, and 7.5% ± 3.3% CD31+ cells, with 2.1 ± 0.7 × 105 cells per cubic centimeter of adipose tissue obtained. Fluorescent CS-1000 (CS-ATM DM Green) labeled 87.0% ± 13.5% of CD34+ progenitor cells compared with 47.8% ± 18.5% of hematopoietic CD45+ cells, with an average of 2.8 ± 2.0 × 1012 19F atoms per cell, determined using nuclear magnetic resonance spectroscopy. The vast majority (92.7% ± 5.0%) of CD31+ cells were also labeled, although most coexpressed CD34. Only 16% ± 22.3% of CD45−/CD31−/CD34− (triple-negative) cells were labeled with CS-ATM DM Green. After induction of cell death by either apoptosis or necrosis, >95% of 19F was released from the cells, indicating that fluorine retention can be used as a surrogate marker for cell survival. Labeled-SVF cells engrafted in a silicone breast phantom could be visualized with a clinical 3-Tesla magnetic resonance imaging scanner at a sensitivity of approximately 2 × 106 cells at a depth of 5 mm. The current protocol can be used to image transplanted SVF cells at clinically relevant cell concentrations in patients. Significance Stromal vascular fraction (SVF) cells harvested from adipose tissue offer great promise in regenerative medicine, but methods to track such cell therapies are needed to ensure correct administration and monitor survival. A clinical protocol

Label-free density difference amplification-based cell sorting.

PubMed

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

2014-11-01

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

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

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.

Label-Free, High Resolution, Multi-Modal Light Microscopy for Discrimination of Live Stem Cell Differentiation Status.

PubMed

Zhang, Jing; Moradi, Emilia; Somekh, Michael G; Mather, Melissa L

2018-01-15

A label-free microscopy method for assessing the differentiation status of stem cells is presented with potential application for characterization of therapeutic stem cell populations. The microscopy system is capable of characterizing live cells based on the use of evanescent wave microscopy and quantitative phase contrast (QPC) microscopy. The capability of the microscopy system is demonstrated by studying the differentiation of live immortalised neonatal mouse neural stem cells over a 15 day time course. Metrics extracted from microscope images are assessed and images compared with results from endpoint immuno-staining studies to illustrate the system's performance. Results demonstrate the potential of the microscopy system as a valuable tool for cell biologists to readily identify the differentiation status of unlabelled live cells.

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

Label-Free, Flow-Imaging Methods for Determination of Cell Concentration and Viability.

PubMed

Sediq, A S; Klem, R; Nejadnik, M R; Meij, P; Jiskoot, Wim

2018-05-30

To investigate the potential of two flow imaging microscopy (FIM) techniques (Micro-Flow Imaging (MFI) and FlowCAM) to determine total cell concentration and cell viability. B-lineage acute lymphoblastic leukemia (B-ALL) cells of 2 different donors were exposed to ambient conditions. Samples were taken at different days and measured with MFI, FlowCAM, hemocytometry and automated cell counting. Dead and live cells from a fresh B-ALL cell suspension were fractionated by flow cytometry in order to derive software filters based on morphological parameters of separate cell populations with MFI and FlowCAM. The filter sets were used to assess cell viability in the measured samples. All techniques gave fairly similar cell concentration values over the whole incubation period. MFI showed to be superior with respect to precision, whereas FlowCAM provided particle images with a higher resolution. Moreover, both FIM methods were able to provide similar results for cell viability as the conventional methods (hemocytometry and automated cell counting). FIM-based methods may be advantageous over conventional cell methods for determining total cell concentration and cell viability, as FIM measures much larger sample volumes, does not require labeling, is less laborious and provides images of individual cells.

Recognition-driven chemical labeling of endogenous proteins in multi-molecular crowding in live cells.

PubMed

Amaike, Kazuma; Tamura, Tomonori; Hamachi, Itaru

2017-11-14

Endogenous protein labeling is one of the most invaluable methods for studying the bona fide functions of proteins in live cells. However, multi-molecular crowding conditions, such as those that occur in live cells, hamper the highly selective chemical labeling of a protein of interest (POI). We herein describe how the efficient coupling of molecular recognition with a chemical reaction is crucial for selective protein labeling. Recognition-driven protein labeling is carried out by a synthetic labeling reagent containing a protein (recognition) ligand, a reporter tag, and a reactive moiety. The molecular recognition of a POI can be used to greatly enhance the reaction kinetics and protein selectivity, even under live cell conditions. In this review, we also briefly discuss how such selective chemical labeling of an endogenous protein can have a variety of applications at the interface of chemistry and biology.

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

NASA Astrophysics Data System (ADS)

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

2009-11-01

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

Quenched substrates for live-cell labeling of SNAP-tagged fusion proteins with improved fluorescent background.

PubMed

Stöhr, Katharina; Siegberg, Daniel; Ehrhard, Tanja; Lymperopoulos, Konstantinos; Öz, Simin; Schulmeister, Sonja; Pfeifer, Andrea C; Bachmann, Julie; Klingmüller, Ursula; Sourjik, Victor; Herten, Dirk-Peter

2010-10-01

Recent developments in fluorescence microscopy raise the demands for bright and photostable fluorescent tags for specific and background free labeling in living cells. Aside from fluorescent proteins and other tagging methods, labeling of SNAP-tagged proteins has become available thereby increasing the pool of potentially applicable fluorescent dyes for specific labeling of proteins. Here, we report on novel conjugates of benzylguanine (BG) which are quenched in their fluorescence and become highly fluorescent upon labeling of the SNAP-tag, the commercial variant of the human O(6)-alkylguanosyltransferase (hAGT). We identified four conjugates showing a strong increase, i.e., >10-fold, in fluorescence intensity upon labeling of SNAP-tag in vitro. Moreover, we screened a subset of nine BG-dye conjugates in living Escherichia coli and found them all suited for labeling of the SNAP-tag. Here, quenched BG-dye conjugates yield a higher specificity due to reduced contribution from excess conjugate to the fluorescence signal. We further extended the application of these conjugates by labeling a SNAP-tag fusion of the Tar chemoreceptor in live E. coli cells and the eukaryotic transcription factor STAT5b in NIH 3T3 mouse fibroblast cells. Aside from the labeling efficiency and specificity in living cells, we discuss possible mechanisms that might be responsible for the changes in fluorescence emission upon labeling of the SNAP-tag, as well as problems we encountered with nonspecific labeling with certain conjugates in eukaryotic cells.

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.

Alteration of Radiosensitivity of Quiescent Cell Populations in Solid Tumors Irradiated with X‐Rays Twice at Various Intervals

PubMed Central

Ono, Koji; Mitsumori, Michihide; Abe, Mitsuyuki

1993-01-01

5‐Bromo‐2′‐deoxyuridine (BUdR) was injected into SCC VII or EMT6/KU tumor‐bearing mice intraperitoneally to label all the proliferating tumor cells. First, the mice were irradiated with X‐rays at a dose of 10 Gy, followed by a dose of 0–20 Gy at 0, 12, 24 or 48 h later. During the interval, no BUdR was injected. Immediately after the second irradiation, the tumors were excised and trypsinized. The micronucleus (MN) frequency in cells without BUdR labeling was determined by means of incubation with cytochalasin‐B (a cytokinesis‐blocker) and immunoftuorescence staining for BUdR. When the tumors were not pretreated with BUdR before the first irradiation, the MN frequency in all tumor cells was determined. To determine the labeling indices of SCC VII and EMT6/KU tumors at the time of the second irradiation, each group also included mice that were continuously administered BUdR until just before the second irradiation using mini‐osmotic pumps which had been implanted subcutaneously 5 days before the first irradiation. The MN frequency of all tumor cell populations obtained immediately after the second irradiation decreased in proportion to the increase in interval time. However, in both tumor systems, the MN frequency of unlabeled cell populations, which could be regarded as quiescent cells in the tumors at the time of the first irradiation, was raised with increase in the interval time. In addition, the labeling index at the second irradiation was higher than that at the first irradiation. These findings support the occurrence of recruitment from quiescent to proliferating state during fractionated irradiation. PMID:8276718

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

PubMed

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

2015-09-15

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

Semi-automated quantification and neuroanatomical mapping of heterogeneous cell populations.

PubMed

Mendez, Oscar A; Potter, Colin J; Valdez, Michael; Bello, Thomas; Trouard, Theodore P; Koshy, Anita A

2018-07-15

Our group studies the interactions between cells of the brain and the neurotropic parasite Toxoplasma gondii. Using an in vivo system that allows us to permanently mark and identify brain cells injected with Toxoplasma protein, we have identified that Toxoplasma-injected neurons (TINs) are heterogeneously distributed throughout the brain. Unfortunately, standard methods to quantify and map heterogeneous cell populations onto a reference brain atlas are time consuming and prone to user bias. We developed a novel MATLAB-based semi-automated quantification and mapping program to allow the rapid and consistent mapping of heterogeneously distributed cells on to the Allen Institute Mouse Brain Atlas. The system uses two-threshold background subtraction to identify and quantify cells of interest. We demonstrate that we reliably quantify and neuroanatomically localize TINs with low intra- or inter-observer variability. In a follow up experiment, we show that specific regions of the mouse brain are enriched with TINs. The procedure we use takes advantage of simple immunohistochemistry labeling techniques, use of a standard microscope with a motorized stage, and low cost computing that can be readily obtained at a research institute. To our knowledge there is no other program that uses such readily available techniques and equipment for mapping heterogeneous populations of cells across the whole mouse brain. The quantification method described here allows reliable visualization, quantification, and mapping of heterogeneous cell populations in immunolabeled sections across whole mouse brains. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

Shrinkage of X cells in the lateral geniculate nucleus after monocular deprivation revealed by FoxP2 labeling.

PubMed

Duffy, Kevin R; Holman, Kaitlyn D; Mitchell, Donald E

2014-05-01

The parallel processing of visual features by distinct neuron populations is a central characteristic of the mammalian visual system. In the A laminae of the cat dorsal lateral geniculate nucleus (dLGN), parallel processing streams originate from two principal neuron types, called X and Y cells. Disruption of visual experience early in life by monocular deprivation has been shown to alter the structure and function of Y cells, but the extent to which deprivation influences X cells remains less clear. A transcription factor, FoxP2, has recently been shown to selectively label X cells in the ferret dLGN and thus provides an opportunity to examine whether monocular deprivation alters the soma size of X cells. In this study, FoxP2 labeling was examined in the dLGN of normal and monocularly deprived cats. The characteristics of neurons labeled for FoxP2 were consistent with FoxP2 being a marker for X cells in the cat dLGN. Monocular deprivation for either a short (7 days) or long (7 weeks) duration did not alter the density of FoxP2-positive neurons between nondeprived and deprived dLGN layers. However, for each deprived animal examined, measurement of the cross-sectional area of FoxP2-positive neurons (X cells) revealed that within deprived layers, X cells were smaller by approximately 20% after 7 days of deprivation, and by approximately 28% after 7 weeks of deprivation. The observed alteration to the cross-sectional area of X cells indicates that perturbation of this major pathway contributes to the functional impairments that develop from monocular deprivation.

Functionalized magnetic-fluorescent hybrid nanoparticles for cell labelling.

PubMed

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

2011-05-01

A facile method of synthesizing 60 nm magnetic-fluorescent core-shell bifunctional nanocomposites with the ability to label cells is presented. Hydrophobic trioctylphosphine oxide (TOPO)-capped CdSe@ZnS quantum dots (QDs) were assembled on polyethyleneimine (PEI)-coated Fe(3)O(4) nanoparticles (MNP). Polyethyleneimine was utilized for the realization of multifunction, including attaching 4 nm TOPO capped CdSe@ZnS quantum dots onto magnetite particles, altering the surface properties of quantum dots from hydrophobic to hydrophilic as well as preventing the formation of large aggregates. Results show that these water-soluble hybrid nanocomposites exhibit good colloidal stability and retain good magnetic and fluorescent properties. Because TOPO-capped QDs are assembled instead of their water-soluble equivalents, the nanocomposites are still highly luminescent with no shift in the PL peak position and present long-term fluorescence stability. Moreover, TAT peptide (GRKKRRQRRRPQ) functionalized hybrid nanoparticles were also studied due to their combined magnetic enrichment and optical detection for cell separation and rapid cell labelling. A cell viability assay revealed good biocompatibility of these hybrid nanoparticles. The potential application of the new magnetic-fluorescent nanocomposites in biological and medicine is demonstrated. © The Royal Society of Chemistry 2011

Selective labelling and eradication of antibiotic-tolerant bacterial populations in Pseudomonas aeruginosa biofilms

PubMed Central

Chua, Song Lin; Yam, Joey Kuok Hoong; Hao, Piliang; Adav, Sunil S.; Salido, May Margarette; Liu, Yang; Givskov, Michael; Sze, Siu Kwan; Tolker-Nielsen, Tim; Yang, Liang

2016-01-01

Drug resistance and tolerance greatly diminish the therapeutic potential of antibiotics against pathogens. Antibiotic tolerance by bacterial biofilms often leads to persistent infections, but its mechanisms are unclear. Here we use a proteomics approach, pulsed stable isotope labelling with amino acids (pulsed-SILAC), to quantify newly expressed proteins in colistin-tolerant subpopulations of Pseudomonas aeruginosa biofilms (colistin is a ‘last-resort' antibiotic against multidrug-resistant Gram-negative pathogens). Migration is essential for the formation of colistin-tolerant biofilm subpopulations, with colistin-tolerant cells using type IV pili to migrate onto the top of the colistin-killed biofilm. The colistin-tolerant cells employ quorum sensing (QS) to initiate the formation of new colistin-tolerant subpopulations, highlighting multicellular behaviour in antibiotic tolerance development. The macrolide erythromycin, which has been previously shown to inhibit the motility and QS of P. aeruginosa, boosts biofilm eradication by colistin. Our work provides insights on the mechanisms underlying the formation of antibiotic-tolerant populations in bacterial biofilms and indicates research avenues for designing more efficient treatments against biofilm-associated infections. PMID:26892159

Selective labelling and eradication of antibiotic-tolerant bacterial populations in Pseudomonas aeruginosa biofilms.

PubMed

Chua, Song Lin; Yam, Joey Kuok Hoong; Hao, Piliang; Adav, Sunil S; Salido, May Margarette; Liu, Yang; Givskov, Michael; Sze, Siu Kwan; Tolker-Nielsen, Tim; Yang, Liang

2016-02-19

Drug resistance and tolerance greatly diminish the therapeutic potential of antibiotics against pathogens. Antibiotic tolerance by bacterial biofilms often leads to persistent infections, but its mechanisms are unclear. Here we use a proteomics approach, pulsed stable isotope labelling with amino acids (pulsed-SILAC), to quantify newly expressed proteins in colistin-tolerant subpopulations of Pseudomonas aeruginosa biofilms (colistin is a 'last-resort' antibiotic against multidrug-resistant Gram-negative pathogens). Migration is essential for the formation of colistin-tolerant biofilm subpopulations, with colistin-tolerant cells using type IV pili to migrate onto the top of the colistin-killed biofilm. The colistin-tolerant cells employ quorum sensing (QS) to initiate the formation of new colistin-tolerant subpopulations, highlighting multicellular behaviour in antibiotic tolerance development. The macrolide erythromycin, which has been previously shown to inhibit the motility and QS of P. aeruginosa, boosts biofilm eradication by colistin. Our work provides insights on the mechanisms underlying the formation of antibiotic-tolerant populations in bacterial biofilms and indicates research avenues for designing more efficient treatments against biofilm-associated infections.

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

PubMed

Fleisig, Helen; Wong, Judy

2012-05-22

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

15N-metabolic labeling for comparative plasma membrane proteomics in Arabidopsis cells.

PubMed

Lanquar, Viviane; Kuhn, Lauriane; Lelièvre, Françoise; Khafif, Mehdi; Espagne, Christelle; Bruley, Christophe; Barbier-Brygoo, Hélène; Garin, Jérôme; Thomine, Sébastien

2007-03-01

An important goal for proteomic studies is the global comparison of proteomes from different genotypes, tissues, or physiological conditions. This has so far been mostly achieved by densitometric comparison of spot intensities after protein separation by 2-DE. However, the physicochemical properties of membrane proteins preclude the use of 2-DE. Here, we describe the use of in vivo labeling by the stable isotope 15N as an alternative approach for comparative membrane proteomic studies in plant cells. We confirm that 15N-metabolic labeling of proteins is possible and efficient in Arabidopsis suspension cells. Quantification of 14N versus 15N MS signals reflects the relative abundance of 14N and 15N proteins in the sample analyzed. We describe the use of 15N-metabolic labeling to perform a partial comparative analysis of Arabidopsis cells following cadmium exposure. By focusing our attention on plasma membrane proteins, we were able to confidently identify proteins showing up to 5-fold regulation compared to unexposed cells. This study provides a proof of principle that 15N-metabolic labeling is a useful technique for comparative membrane proteome studies.

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

PubMed Central

Cao, Mingzhu; Yeung, William S.B.

2015-01-01

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

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

PubMed

Lu, Ting; Lin, Zongwei; Ren, Jianwei; Yao, Peng; Wang, Xiaowei; Wang, Zhe; Zhang, Qunye

2016-01-01

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

Magnetic Resonance Imaging of Iron Oxide-Labeled Human Embryonic Stem Cell-Derived Cardiac Progenitors.

PubMed

Skelton, Rhys J P; Khoja, Suhail; Almeida, Shone; Rapacchi, Stanislas; Han, Fei; Engel, James; Zhao, Peng; Hu, Peng; Stanley, Edouard G; Elefanty, Andrew G; Kwon, Murray; Elliott, David A; Ardehali, Reza

2016-01-01

Given the limited regenerative capacity of the heart, cellular therapy with stem cell-derived cardiac cells could be a potential treatment for patients with heart disease. However, reliable imaging techniques to longitudinally assess engraftment of the transplanted cells are scant. To address this issue, we used ferumoxytol as a labeling agent of human embryonic stem cell-derived cardiac progenitor cells (hESC-CPCs) to facilitate tracking by magnetic resonance imaging (MRI) in a large animal model. Differentiating hESCs were exposed to ferumoxytol at different time points and varying concentrations. We determined that treatment with ferumoxytol at 300 μg/ml on day 0 of cardiac differentiation offered adequate cell viability and signal intensity for MRI detection without compromising further differentiation into definitive cardiac lineages. Labeled hESC-CPCs were transplanted by open surgical methods into the left ventricular free wall of uninjured pig hearts and imaged both ex vivo and in vivo. Comprehensive T2*-weighted images were obtained immediately after transplantation and 40 days later before termination. The localization and dispersion of labeled cells could be effectively imaged and tracked at days 0 and 40 by MRI. Thus, under the described conditions, ferumoxytol can be used as a long-term, differentiation-neutral cell-labeling agent to track transplanted hESC-CPCs in vivo using MRI. The development of a safe and reproducible in vivo imaging technique to track the fate of transplanted human embryonic stem cell-derived cardiac progenitor cells (hESC-CPCs) is a necessary step to clinical translation. An iron oxide nanoparticle (ferumoxytol)-based approach was used for cell labeling and subsequent in vivo magnetic resonance imaging monitoring of hESC-CPCs transplanted into uninjured pig hearts. The present results demonstrate the use of ferumoxytol labeling and imaging techniques in tracking the location and dispersion of cell grafts, highlighting its

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

PubMed Central

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

2016-01-01

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

Systematically labeling developmental stage-specific genes for the study of pancreatic β-cell differentiation from human embryonic stem cells.

PubMed

Liu, Haisong; Yang, Huan; Zhu, Dicong; Sui, Xin; Li, Juan; Liang, Zhen; Xu, Lei; Chen, Zeyu; Yao, Anzhi; Zhang, Long; Zhang, Xi; Yi, Xing; Liu, Meng; Xu, Shiqing; Zhang, Wenjian; Lin, Hua; Xie, Lan; Lou, Jinning; Zhang, Yong; Xi, Jianzhong; Deng, Hongkui

2014-10-01

The applications of human pluripotent stem cell (hPSC)-derived cells in regenerative medicine has encountered a long-standing challenge: how can we efficiently obtain mature cell types from hPSCs? Attempts to address this problem are hindered by the complexity of controlling cell fate commitment and the lack of sufficient developmental knowledge for guiding hPSC differentiation. Here, we developed a systematic strategy to study hPSC differentiation by labeling sequential developmental genes to encompass the major developmental stages, using the directed differentiation of pancreatic β cells from hPSCs as a model. We therefore generated a large panel of pancreas-specific mono- and dual-reporter cell lines. With this unique platform, we visualized the kinetics of the entire differentiation process in real time for the first time by monitoring the expression dynamics of the reporter genes, identified desired cell populations at each differentiation stage and demonstrated the ability to isolate these cell populations for further characterization. We further revealed the expression profiles of isolated NGN3-eGFP(+) cells by RNA sequencing and identified sushi domain-containing 2 (SUSD2) as a novel surface protein that enriches for pancreatic endocrine progenitors and early endocrine cells both in human embryonic stem cells (hESC)-derived pancreatic cells and in the developing human pancreas. Moreover, we captured a series of cell fate transition events in real time, identified multiple cell subpopulations and unveiled their distinct gene expression profiles, among heterogeneous progenitors for the first time using our dual reporter hESC lines. The exploration of this platform and our new findings will pave the way to obtain mature β cells in vitro.

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

PubMed

Ackerman, G A; Wolken, K W

1981-10-01

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

Radioisotopic Method for Measuring Cell Division Rates of Individual Species of Diatoms from Natural Populations †

PubMed Central

Rivkin, Richard B.

1986-01-01

Silicon is an essential element for diatom frustule synthesis and is usually taken up only by dividing cells. With 68Ge, a radioactive analog of Si, the cell cycle marker event of frustule formation was identified for individual species of diatom. The frequency of cells within a population undergoing this division event was estimated, and the cell division rate was calculated. In laboratory cultures, these rates of cell division and those calculated from changes in cell numbers were similar. By dual labeling with 68Ge(OH)4 and NaH14CO3, rates of cell division and photosynthesis were coincidently measured for diatoms both in laboratory cultures and when isolated from natural populations in estuarine, offshore, and polar environments. These techniques permit the coupling between photosynthesis and cell division to be examined in situ for individual species of diatom. PMID:16347039

Photoluminescent diamond nanoparticles for cell labeling: study of the uptake mechanism in mammalian cells.

PubMed

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

2009-12-22

Diamond nanoparticles (nanodiamonds) have been recently proposed as new labels for cellular imaging. For small nanodiamonds (size <40 nm), resonant laser scattering and Raman scattering cross sections are too small to allow single nanoparticle observation. Nanodiamonds can, however, be rendered photoluminescent with a perfect photostability at room temperature. Such a remarkable property allows easier single-particle tracking over long time scales. In this work, we use photoluminescent nanodiamonds of size <50 nm for intracellular labeling and investigate the mechanism of their uptake by living cells. By blocking selectively different uptake processes, we show that nanodiamonds enter cells mainly by endocytosis, and converging data indicate that it is clathrin-mediated. We also examine nanodiamond intracellular localization in endocytic vesicles using immunofluorescence and transmission electron microscopy. We find a high degree of colocalization between vesicles and the biggest nanoparticles or aggregates, while the smallest particles appear free in the cytosol. Our results pave the way for the use of photoluminescent nanodiamonds in targeted intracellular labeling or biomolecule delivery.

18F-FDG labeling of mesenchymal stem cells and multipotent adult progenitor cells for PET imaging: effects on ultrastructure and differentiation capacity.

PubMed

Wolfs, Esther; Struys, Tom; Notelaers, Tineke; Roberts, Scott J; Sohni, Abhishek; Bormans, Guy; Van Laere, Koen; Luyten, Frank P; Gheysens, Olivier; Lambrichts, Ivo; Verfaillie, Catherine M; Deroose, Christophe M

2013-03-01

Because of their extended differentiation capacity, stem cells have gained great interest in the field of regenerative medicine. For the development of therapeutic strategies, more knowledge on the in vivo fate of these cells has to be acquired. Therefore, stem cells can be labeled with radioactive tracer molecules such as (18)F-FDG, a positron-emitting glucose analog that is taken up and metabolically trapped by the cells. The aim of this study was to optimize the radioactive labeling of mesenchymal stem cells (MSCs) and multipotent adult progenitor cells (MAPCs) in vitro with (18)F-FDG and to investigate the potential radiotoxic effects of this labeling procedure with a range of techniques, including transmission electron microscopy (TEM). Mouse MSCs and rat MAPCs were used for (18)F-FDG uptake kinetics and tracer retention studies. Cell metabolic activity, proliferation, differentiation and ultrastructural changes after labeling were evaluated using an Alamar Blue reagent, doubling time calculations and quantitative TEM, respectively. Additionally, mice were injected with MSCs and MAPCs prelabeled with (18)F-FDG, and stem cell biodistribution was investigated using small-animal PET. The optimal incubation period for (18)F-FDG uptake was 60 min. Significant early tracer washout was observed, with approximately 30%-40% of the tracer being retained inside the cells 3 h after labeling. Cell viability, proliferation, and differentiation capacity were not severely affected by (18)F-FDG labeling. No major changes at the ultrastructural level, considering mitochondrial length, lysosome size, the number of lysosomes, the number of vacuoles, and the average rough endoplasmic reticulum width, were observed with TEM. Small-animal PET experiments with radiolabeled MAPCs and MSCs injected intravenously in mice showed a predominant accumulation in the lungs and a substantial elution of (18)F-FDG from the cells. MSCs and MAPCs can be successfully labeled with (18)F-FDG for

In Silico Labeling: Predicting Fluorescent Labels in Unlabeled Images.

PubMed

Christiansen, Eric M; Yang, Samuel J; Ando, D Michael; Javaherian, Ashkan; Skibinski, Gaia; Lipnick, Scott; Mount, Elliot; O'Neil, Alison; Shah, Kevan; Lee, Alicia K; Goyal, Piyush; Fedus, William; Poplin, Ryan; Esteva, Andre; Berndl, Marc; Rubin, Lee L; Nelson, Philip; Finkbeiner, Steven

2018-04-19

Microscopy is a central method in life sciences. Many popular methods, such as antibody labeling, are used to add physical fluorescent labels to specific cellular constituents. However, these approaches have significant drawbacks, including inconsistency; limitations in the number of simultaneous labels because of spectral overlap; and necessary perturbations of the experiment, such as fixing the cells, to generate the measurement. Here, we show that a computational machine-learning approach, which we call "in silico labeling" (ISL), reliably predicts some fluorescent labels from transmitted-light images of unlabeled fixed or live biological samples. ISL predicts a range of labels, such as those for nuclei, cell type (e.g., neural), and cell state (e.g., cell death). Because prediction happens in silico, the method is consistent, is not limited by spectral overlap, and does not disturb the experiment. ISL generates biological measurements that would otherwise be problematic or impossible to acquire. Copyright © 2018 Elsevier Inc. All rights reserved.

A rapid and fluorogenic TMP-AcBOPDIPY probe for covalent labeling of proteins in live cells.

PubMed

Liu, Wei; Li, Fu; Chen, Xi; Hou, Jian; Yi, Long; Wu, Yao-Wen

2014-03-26

Protein labeling is enormously useful for characterizing protein function in cells and organisms. Chemical tagging methods have emerged as a new generation protein labeling strategy in live cells. Here we have developed a novel and versatile TMP-AcBOPDIPY probe for selective and turn-on labeling of proteins in live cells. A small monomeric tag, E. coli dihydrofolate reductase (eDHFR), was rationally designed to introduce a cysteine in the vicinity of the ligand binding site. Trimethoprim (TMP) that specifically binds to eDHFR was linked to the BOPDIPY fluorophore containing a mildly thiol-reactive acrylamide group. TMP-AcBOPDIPY rapidly labeled engineered eDHFR tags via a reaction termed affinity conjugation (a half-life of ca. 2 min), which is one of the top fast chemical probes for protein labeling. The probe displays 2-fold fluorescence enhancement upon labeling of proteins. We showed that the probe specifically labeled intracellular proteins in live cells without and with washing out the dye. We demonstrated its utility in visualizing intracellular processes by fluorescence-lifetime imaging microscopy (FLIM) measurements.

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 specifications...

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 specifications...

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 specifications...

Connective Tissue Growth Factor reporter mice label a subpopulation of mesenchymal progenitor cells that reside in the trabecular bone region.

PubMed

Wang, Wen; Strecker, Sara; Liu, Yaling; Wang, Liping; Assanah, Fayekah; Smith, Spenser; Maye, Peter

2015-02-01

Few gene markers selectively identify mesenchymal progenitor cells inside the bone marrow. We have investigated a cell population located in the mouse bone marrow labeled by Connective Tissue Growth Factor reporter expression (CTGF-EGFP). Bone marrow flushed from CTGF reporter mice yielded an EGFP+ stromal cell population. Interestingly, the percentage of stromal cells retaining CTGF reporter expression decreased with age in vivo and was half the frequency in females compared to males. In culture, CTGF reporter expression and endogenous CTGF expression marked the same cell types as those labeled using Twist2-Cre and Osterix-Cre fate mapping approaches, which previously had been shown to identify mesenchymal progenitors in vitro. Consistent with this past work, sorted CTGF+ cells displayed an ability to differentiate into osteoblasts, chondrocytes, and adipocytes in vitro and into osteoblast, adipocyte, and stromal cell lineages after transplantation into a parietal bone defect. In vivo examination of CTGF reporter expression in bone tissue sections revealed that it marked cells highly localized to the trabecular bone region and was not expressed in the perichondrium or periosteum. Mesenchymal cells retaining high CTGF reporter expression were adjacent to, but distinct from mature osteoblasts lining bone surfaces and endothelial cells forming the vascular sinuses. Comparison of CTGF and Osterix reporter expression in bone tissue sections indicated an inverse correlation between the strength of CTGF expression and osteoblast maturation. Down-regulation of CTGF reporter expression also occurred during in vitro osteogenic differentiation. Collectively, our studies indicate that CTGF reporter mice selectively identify a subpopulation of bone marrow mesenchymal progenitor cells that reside in the trabecular bone region. Copyright © 2014 Elsevier Inc. All rights reserved.

Connective Tissue Growth Factor Reporter Mice Label a Subpopulation of Mesenchymal Progenitor Cells that Reside in the Trabecular Bone Region

PubMed Central

Wang, Wen; Strecker, Sara; Liu, Yaling; Wang, Liping; Assanah, Fayekah; Smith, Spenser; Maye, Peter

2014-01-01

Few gene markers selectively identify mesenchymal progenitor cells inside the bone marrow. We have investigated a cell population located in the mouse bone marrow labeled by Connective Tissue Growth Factor reporter expression (CTGF-EGFP). Bone marrow flushed from CTGF reporter mice yielded an EGFP+ stromal cell population. Interestingly, the percentage of stromal cells retaining CTGF reporter expression decreased with age in vivo and was half the frequency in females compared to males. In culture, CTGF reporter expression and endogenous CTGF expression marked the same cell types as those labeled using Twist2-Cre and Osterix-Cre fate mapping approaches, which previously has been shown to identify mesenchymal progenitors in vitro. Consistent with this past work, sorted CTGF+ cells displayed an ability to differentiate into osteoblasts, chondrocytes, and adipocytes in vitro and into osteoblast, adipocyte, and stromal cell lineages after transplantation into a parietal bone defect. In vivo examination of CTGF reporter expression in bone tissue sections revealed it marked cells highly localized to the trabecular bone region and was not expressed in the perichondrium or periosteum. Mesenchymal cells retaining high CTGF reporter expression were adjacent to, but distinct from mature osteoblasts lining bone surfaces and endothelial cells forming the vascular sinuses. Comparison of CTGF and Osterix reporter expression in bone tissue sections indicated an inverse correlation between the strength of CTGF expression and osteoblast maturation. Down-regulation of CTGF reporter expression also occurred during in vitro osteogenic differentiation. Collectively, our studies indicate that CTGF reporter mice selectively identify a subpopulation of bone marrow mesenchymal progenitor cells that reside in the trabecular bone region. PMID:25464947

Label-Free, High-Throughput Purification of Satellite Cells Using Microfluidic Inertial Separation.

PubMed

Syverud, Brian C; Lin, Eric; Nagrath, Sunitha; Larkin, Lisa M

2018-01-01

Skeletal muscle satellite cells have tremendous therapeutic potential in cell therapy or skeletal muscle tissue engineering. Obtaining a sufficiently pure satellite cell population, however, presents a significant challenge. We hypothesized that size differences between satellite cells and fibroblasts, two primary cell types obtained from skeletal muscle dissociation, would allow for label-free, inertial separation in a microfluidic device, termed a "Labyrinth," and that these purified satellite cells could be used to engineer skeletal muscle. Throughout tissue fabrication, Labyrinth-purified cells were compared with unsorted controls to assess the efficiency of this novel sorting process and to examine potential improvements in myogenic proliferation, differentiation, and tissue function. Immediately after dissociation and Labyrinth sorting, cells were immunostained to identify myogenic cells and fibroblast progenitors. Remaining cells were cultured for 14 days to form a confluent monolayer that was induced to delaminate and was captured as a 3D skeletal muscle construct. During monolayer development, myogenic proliferation (BrdU assay on Day 4), differentiation and myotube fusion index (α-actinin on Day 11), and myotube structural development (light microscopy on Day 14) were assessed. Isometric tetanic force production was measured in 3D constructs on Day 16. Immediately following sorting, unsorted cells exhibited a myogenic purity of 39.9% ± 3.99%, and this purity was enriched approximately two-fold to 75.5% ± 1.59% by microfluidic separation. The BrdU assay on Day 4 similarly showed significantly enhanced myogenic proliferation: in unsorted controls 47.0% ± 2.77% of proliferating cells were myogenic, in comparison to 61.7% ± 2.55% following purification. Myogenic differentiation and fusion, assessed by fusion index quantification, showed improvement from 82.7% ± 3.74% in control to 92.3% ± 2.04% in the purified cell population

The long-term label retaining population of the renal papilla arises through divergent regional growth of the kidney.

PubMed

Adams, Derek C; Oxburgh, Leif

2009-09-01

Long-term pulse chase experiments previously identified a sizable population of BrdU-retaining cells within the renal papilla. The origin of these cells has been unclear, and in this work we test the hypothesis that they become quiescent early during the course of kidney development and organ growth. Indeed, we find that BrdU-retaining cells of the papilla can be labeled only by pulsing with BrdU from embryonic (E) day 11.25 to postnatal (P) day 7, the approximate period of kidney development in the mouse. BrdU signal in the cortex and outer medulla is rapidly diluted by cellular proliferation during embryonic development and juvenile growth, whereas cells within the papilla differentiate and exit the cell cycle during organogenesis. Indeed, by E17.5, little or no active proliferation can be seen in the distal papilla, indicating maturation of this structure in a distal-to-proximal manner during organogenesis. We conclude that BrdU-retaining cells of the papilla represent a population of cells that quiesce during embryonic development and localize within a region of the kidney that matures early. We therefore propose that selective papillary retention of BrdU arises through a combination of regionalized slowing of, and exit from, the cell cycle within the papilla during the period of ongoing kidney development, and extensive proliferative growth of the juvenile kidney resulting in dilution of BrdU below the detection level in extra-papillary regions.

Capture and detection of Staphylococcus aureus with dual labeled aptamers to cell surface components.

PubMed

Ramlal, Shylaja; Mondal, Bhairab; Lavu, Padma Sudharani; N, Bhavanashri; Kingston, Joseph

2018-01-16

In the present study, a high throughput whole cell SELEX method has been applied successfully in selecting specific aptamers against whole cells of Staphylococcus aureus, a potent food poisoning bacterium. A total ten rounds of SELEX and three rounds of intermittent counter SELEX, was performed to obtain specific aptamers. Obtained oligonucleotide pool were cloned, sequenced and then grouped into different families based on their primary sequence homology and secondary structure similarity. FITC labeled sequences from different families were selected for further characterization via flow cytometry analysis. The dissociation constant (K d ) values of specific and higher binders ranged from 34 to 128nM. Binding assays to assess the selectivity of aptamer RAB10, RAB 20, RAB 28 and RAB 35 demonstrated high affinity against S. aureus and low binding affinity for other bacteria. To demonstrate the potential use of the aptamer a sensitive dual labeled sandwich detection system was developed using aptamer RAB10 and RAB 35 with a detection limit of 10 2 CFU/mL. Furthermore detection from mixed cell population and spiked sample emphasized the robustness as well as applicability of the developed method. Altogether, the established assay could be a reliable detection tool for the routine investigation of Staphylococcus aureus in samples from food and clinical sources. Copyright © 2017. Published by Elsevier B.V.

Breast cancer cells synchronous labeling and separation based on aptamer and fluorescence-magnetic silica nanoparticles

NASA Astrophysics Data System (ADS)

Wang, Qiu-Yue; Huang, Wei; Jiang, Xing-Lin; Kang, Yan-Jun

2018-01-01

In this work, an efficient method based on biotin-labeled aptamer and streptavidin-conjugated fluorescence-magnetic silica nanoprobes (FITC@Fe3O4@SiNPs-SA) has been established for human breast carcinoma MCF-7 cells synchronous labeling and separation. Carboxyl-modified fluorescence-magnetic silica nanoparticles (FITC@Fe3O4@SiNPs-COOH) were first synthesized using the Stöber method. Streptavidin (SA) was then conjugated to the surface of FITC@Fe3O4@SiNPs-COOH. The MCF-7 cell suspension was incubated with biotin-labeled MUC-1 aptamer. After centrifugation and washing, the cells were then treated with FITC@Fe3O4@SiNPs-SA. Afterwards, the mixtures were separated by a magnet. The cell-probe conjugates were then imaged using fluorescent microscopy. The results show that the MUC-1 aptamer could recognize and bind to the targeted cells with high affinity and specificity, indicating the prepared FITC@Fe3O4@SiNPs-SA with great photostability and superparamagnetism could be applied effectively in labeling and separation for MCF-7 cell in suspension synchronously. In addition, the feasibility of MCF-7 cells detection in peripheral blood was assessed. The results indicate that the method above is also applicable for cancer cells synchronous labeling and separation in complex biological system.

Live-cell MRI with xenon hyper-CEST biosensors targeted to metabolically labeled cell-surface glycans.

PubMed

Witte, Christopher; Martos, Vera; Rose, Honor May; Reinke, Stefan; Klippel, Stefan; Schröder, Leif; Hackenberger, Christian P R

2015-02-23

The targeting of metabolically labeled glycans with conventional MRI contrast agents has proved elusive. In this work, which further expands the utility of xenon Hyper-CEST biosensors in cell experiments, we present the first successful molecular imaging of such glycans using MRI. Xenon Hyper-CEST biosensors are a novel class of MRI contrast agents with very high sensitivity. We designed a multimodal biosensor for both fluorescent and xenon MRI detection that is targeted to metabolically labeled sialic acid through bioorthogonal chemistry. Through the use of a state of the art live-cell bioreactor, it was demonstrated that xenon MRI biosensors can be used to image cell-surface glycans at nanomolar concentrations. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Labeling and Magnetic Resonance Imaging of Exosomes Isolated from Adipose Stem Cells.

PubMed

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

2017-06-19

Adipose stem cells (ASC) represent a promising therapeutic approach for neurodegenerative diseases. Most biological effects of ASC are probably mediated by extracellular vesicles, such as exosomes, which influence the surrounding cells. Current development of exosome therapies requires efficient and noninvasive methods to localize, monitor, and track the exosomes. Among imaging methods used for this purpose, magnetic resonance imaging (MRI) has advantages: high spatial resolution, rapid in vivo acquisition, and radiation-free operation. To be detectable with MRI, exosomes must be labeled with MR contrast agents, such as ultra-small superparamagnetic iron oxide nanoparticles (USPIO). Here, we set up an innovative approach for exosome labeling that preserves their morphology and physiological characteristics. We show that by labeling ASC with USPIO before extraction of nanovesicles, the isolated exosomes retain nanoparticles and can be visualized by MRI. The current work aims at validating this novel USPIO-based exosome labeling method by monitoring the efficiency of the labeling with MRI both in ASC and in exosomes. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Knowledge, attitudes and potential response to menu labelling in an urban public health clinic population.

PubMed

Piron, Jennifer; Smith, Lisa V; Simon, Paul; Cummings, Patricia L; Kuo, Tony

2010-04-01

The present study examines the receptivity to and potential effects of menu labelling on food choices of low-income and minority individuals - a group often at disproportionate risk for preventable, lifestyle-related health conditions (e.g. obesity, diabetes and CVD). We conducted a cross-sectional survey to examine the knowledge, attitudes and potential response to menu labelling in an urban public health clinic population. United States. A total of 639 clinic patients were recruited in the waiting rooms of six, large public health centres in Los Angeles County (2007-2008). These centres provide services to a largely uninsured or under-insured, low-income, Latino and African-American population. Among those approached and who met eligibility criteria, 88 % completed the survey. Of the 639 respondents, 55 % were overweight or obese based on self-reported heights and weights; 74 % reported visiting a fast food restaurant at least once in the past year, including 22 % at least once a week; 93 % thought that calorie information was 'important'; and 86 % thought that restaurants should be required to post calorie information on their menu boards. In multivariate analyses, respondents who were obese, female, Latino and supportive of calorie postings were more likely than others to report that they would choose food and beverages with lower calories as a result of menu labelling. These findings suggest that clinic patients are receptive to this population-based strategy and that they would be inclined to change their food selections in response to menu labelling.

Super-Chelators for Advanced Protein Labeling in Living Cells.

PubMed

Gatterdam, Karl; Joest, Eike F; Dietz, Marina S; Heilemann, Mike; Tampé, Robert

2018-05-14

Live-cell labeling, super-resolution microscopy, single-molecule applications, protein localization, or chemically induced assembly are emerging approaches, which require specific and very small interaction pairs. The minimal disturbance of protein function is essential to derive unbiased insights into cellular processes. Herein, we define a new class of hexavalent N-nitrilotriacetic acid (hexaNTA) chelators, displaying the highest affinity and stability of all NTA-based small interaction pairs described so far. Coupled to bright organic fluorophores with fine-tuned photophysical properties, the super-chelator probes were delivered into human cells by chemically gated nanopores. These super-chelators permit kinetic profiling, multiplexed labeling of His 6 - and His 12 -tagged proteins as well as single-molecule-based super-resolution imaging. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Survival of mature mouse olfactory sensory neurons labeled genetically perinatally.

PubMed

Holl, Anna-Maria

2018-04-01

The main olfactory epithelium (MOE) of an adult mouse harbors a few million mature olfactory sensory neurons (OSNs), which are traditionally defined as mature by their expression of the olfactory marker protein (OMP). Mature OSNs differentiate in situ from stem cells at the base of the MOE. The consensus view is that mature OSNs have a defined lifespan and then undergo programmed cell death, and that the adult MOE maintains homeostasis by generating new mature OSNs from stem cells. But there is also evidence for mature OSNs that are long-lived. Thus far modern genetic tools have not been applied to quantify survival of a population of OSNs that are mature at a given point in time. Here, a genetic strategy was developed to label irreversibly OMP-expressing OSNs in mice. A gene-targeted OMP-CreERT2 strain was generated in which mature OSNs express an enzymatically inactive version of the Cre recombinase. The fusion protein CreERT2 becomes transiently active when exposed to tamoxifen, and in the presence of a Cre reporter in the genome such as tdRFP, CreERT2-expressing cells become irreversibly labeled. A cohort of mice was generated with the same day of birth by in vitro fertilization and embryo transfer, and injected tamoxifen in their mothers at E18.5 of gestation. I counted RFP immunoreactive cells in the MOE and vomeronasal organ of 36 tamoxifen-exposed OMP-CreERT2 × tdRFP mice from 7 age groups: postnatal day (PD)1.5, PD3.5, PD6.5, 3 weeks, 9 weeks, 6 months, and 12 months. Approximately 7.8% of perinatally labeled cells remain at 12 months, confirming that some mature OSNs are indeed long-lived. The survival curve of the population of perinatally labeled MOE cells can be modeled with a mean half-life of 26 days for the population as a whole, excluding the long-lived cells. Copyright © 2018 The Author. Published by Elsevier Inc. All rights reserved.

Biological Characteristics of Fluorescent Superparamagnetic Iron Oxide Labeled Human Dental Pulp Stem Cells

PubMed Central

Li, Ming-wei; Bai, Yu; Guo, Hui-hui

2017-01-01

Tracking transplanted stem cells is necessary to clarify cellular properties and improve transplantation success. In this study, we investigate the effects of fluorescent superparamagnetic iron oxide particles (SPIO) (Molday ION Rhodamine-B™, MIRB) on biological properties of human dental pulp stem cells (hDPSCs) and monitor hDPSCs in vitro and in vivo using magnetic resonance imaging (MRI). Morphological analysis showed that intracellular MIRB particles were distributed in the cytoplasm surrounding the nuclei of hDPSCs. 12.5–100 μg/mL MIRB all resulted in 100% labeling efficiency. MTT showed that 12.5–50 μg/mL MIRB could promote cell proliferation and MIRB over 100 μg/mL exhibited toxic effect on hDPSCs. In vitro MRI showed that 1 × 106 cells labeled with various concentrations of MIRB (12.5–100 μg/mL) could be visualized. In vivo MRI showed that transplanted cells could be clearly visualized up to 60 days after transplantation. These results suggest that 12.5–50 μg/mL MIRB is a safe range for labeling hDPSCs. MIRB labeled hDPSCs cell can be visualized by MRI in vitro and in vivo. These data demonstrate that MIRB is a promising candidate for hDPSCs tracking in hDPSCs based dental pulp regeneration therapy. PMID:28298928

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

PubMed

Salamon, J; Wicklein, D; Didié, M; Lange, C; Schumacher, U; Adam, G; Peldschus, K

2014-04-01

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

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

Development and testing of a new disposable sterile device for labelling white blood cells.

PubMed

Signore, A; Glaudemans, A W J M; Malviya, G; Lazzeri, E; Prandini, N; Viglietti, A L; De Vries, E F J; Dierckx, R A J O

2012-08-01

White blood cell (WBC) labelling requires isolation of cells from patient's blood under sterile conditions using sterile materials, buffers and disposables under good manufacturing practice (GMP) conditions. Till now, this limited the use of white blood cell scintigraphy (WBC-S) only to well equipped laboratories with trained personnel. We invented, developed and tested a disposable, sterile, closed device for blood manipulation, WBC purification and radionuclide labelling without exposing patient's blood and the operator to contamination risks. This device prototype and a final industrialized device (Leukokit®) were tested for WBC labelling and compared to standard procedure. Leukokit® was also tested in an international multi-centre study for easiness of WBC purification and labelling. On the device prototype we tested in parallel, with blood samples from 7 volunteers, the labelling procedure compared to the standard procedure of the International Society of Radiolabeled Blood Elements (ISORBE) consensus protocol with respect to cell recovery, labelling efficiency (LE), cell viability (Trypan Blue test) and sterility (haemoculture). On the final Leukokit® we tested the biocompatibility of all components, and again the LE, erythro-sedimentation rate, cell viability, sterility and apyrogenicity. ACD-A, HES and PBS provided by Leukokit® were also compared to Heparin, Dextran and autologous plasma, respectively. In 4 samples, we tested the chemotactic activity of purified WBC against 1 mg/ml of lipopolysaccharide (LPS) and chemotaxis of 99mTc-HMPAO-labelled WBC (925 MBq) was compared to that of unlabelled cells. For the multi-centre study, 70 labellings were performed with the Leukokit® by 9 expert operators and 3 beginners from five centers using blood from both patients and volunteers. Finally, Media-Fill tests were performed by 3 operators on two different days (11 procedures) by replacing blood and kit reagents with bacterial culture media (Tryptic Soy Broth

Transformation of cell-derived microparticles into quantum-dot-labeled nanovectors for antitumor siRNA delivery.

PubMed

Chen, Gang; Zhu, Jun-Yi; Zhang, Zhi-Ling; Zhang, Wei; Ren, Jian-Gang; Wu, Min; Hong, Zheng-Yuan; Lv, Cheng; Pang, Dai-Wen; Zhao, Yi-Fang

2015-01-12

Cell-derived microparticles (MPs) have been recently recognized as critical intercellular information conveyors. However, further understanding of their biological behavior and potential application has been hampered by the limitations of current labeling techniques. Herein, a universal donor-cell-assisted membrane biotinylation strategy was proposed for labeling MPs by skillfully utilizing the natural membrane phospholipid exchange of their donor cells. This innovative strategy conveniently led to specific, efficient, reproducible, and biocompatible quantum dot (QD) labeling of MPs, thereby reliably conferring valuable traceability on MPs. By further loading with small interference RNA, QD-labeled MPs that had inherent cell-targeting and biomolecule-conveying ability were successfully employed for combined bioimaging and tumor-targeted therapy. This study provides the first reliable and biofriendly strategy for transforming biogenic MPs into functionalized nanovectors. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Labeling of neuronal differentiation and neuron cells with biocompatible fluorescent nanodiamonds

PubMed Central

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

2014-01-01

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. PMID:24830447

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

PubMed

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

2014-05-16

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.

Response of the oligodendrocyte progenitor cell population (defined by NG2 labelling) to demyelination of the adult spinal cord.

PubMed

Keirstead, H S; Levine, J M; Blakemore, W F

1998-02-01

Elucidation of the response of oligodendrocyte progenitor cell populations to demyelination in the adult central nervous system (CNS) is critical to understanding why remyelination fails in multiple sclerosis. Using the anti-NG2 monoclonal antibody to identify oligodendrocyte progenitor cells, we have documented their response to antibody-induced demyelination in the dorsal column of the adult rat spinal cord. The number of NG2+ cells in the vicinity of demyelinated lesions increased by 72% over the course of 3 days following the onset of demyelination. This increase in NG2+ cell numbers did not reflect a nonspecific staining of reactive cells, as GFAP, OX-42, and Rip antibodies did not co-localise with NG2 + cells in double immunostained tissue sections. NG2 + cells incorporated BrdU 48-72 h following the onset of demyelination. After the onset of remyelination (10-14 days), the number of NG2+ cells decreased to 46% of control levels and remained consistently low for 2 months. When spinal cords were exposed to 40 Grays of x-irradiation prior to demyelination, the number of NG2+ cells decreased to 48% of control levels by 3 days following the onset of demyelination and remained unchanged at 3 weeks. Since 40 Grays of x-irradiation kills dividing cells, these studies illustrate a responsive and nonresponsive NG2+ cell population following demyelination in the adult spinal cord and suggest that the responsive NG2+ cell population does not renew itself.

Label-free screening of niche-to-niche variation in satellite stem cells using functionalized pores

NASA Astrophysics Data System (ADS)

Chapman, Matthew R.; Balakrishnan, Karthik; Conboy, Michael J.; Mohanty, Swomitra; Jabart, Eric; Huang, Haiyan; Hack, James; Conboy, Irina M.; Sohn, Lydia L.

2012-02-01

Combinations of surface markers are currently used to identify muscle satellite cells. Using pores functionalized with specific antibodies and measuring the transit time of cells passing through these pores, we discovered remarkable heterogeneity in the expression of these markers in muscle (satellite) stem cells that reside in different single myofibers. Microniche-specific variation in stem cells of the same organ has not been previously described, as bulk analysis does not discriminate between separate myofibers or even separate hind-leg muscle groups. We found a significant population of Sca-1+ satellite cells that form myotubes, thereby demonstrating the myogenic potential of Sca-1+ cells, which are currently excluded in bulk sorting. Finally, using our label-free pore screening technique, we have been able to quantify directly surface expression of Notch1 without activation of the Notch pathway. We show for the first time Notch1-expression heterogeneity in unactivated satellite cells. The discovery of fiber-to-fiber variations prompts new research into the reasons for such diversity in muscle stem cells.

Menu labeling perception and health behaviors among immigrant and US born minority populations: assessment in two Los Angeles public markets.

PubMed

Vargas-Bustamante, Arturo

2013-01-01

To analyze menu labeling perception and food choices/health behaviors in two Los Angeles public markets. Labels with food caloric content were displayed in the food court of one of these markets. Bivarate means analyses compared the surveyed population by market and by nativity status. The main predictors of menu-labeling influence were identified in the sample from the market that displayed labels. A separate analysis investigated food choices/health behaviors among immigrant cohorts by time of US residence. Reading labels when shopping was one of the main predictors associated with menu labeling influence. Longer-stayed immigrants were more likely to afford "balanced meals", but they were also more likely to eat in fast food restaurants and less likely to engage into moderate/intense physical activity. While nativity was not a significant predictor of menu labeling influence on food choices, our findings suggest food choices/behaviors convergence among immigrant and US-born populations.

Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells.

PubMed

Lopata, Anna; Hughes, Ruth; Tiede, Christian; Heissler, Sarah M; Sellers, James R; Knight, Peter J; Tomlinson, Darren; Peckham, Michelle

2018-04-26

Imaging the actin cytoskeleton in cells uses a wide range of approaches. Typically, a fluorescent derivative of the small cyclic peptide phalloidin is used to image F-actin in fixed cells. Lifeact and F-tractin are popular for imaging the cytoskeleton in live cells. Here we characterised novel affinity reagents called Affimers that specifically bind to F-actin in vitro to determine if they are suitable alternatives as eGFP-fusion proteins, to label actin in live cells, or for labeling F-actin in fixed cells. In vitro experiments showed that 3 out of the 4 Affimers (Affimers 6, 14 and 24) tested bind tightly to purified F-actin, and appear to have overlapping binding sites. As eGFP-fusion proteins, the same 3 Affimers label F-actin in live cells. FRAP experiments suggest that eGFP-Affimer 6 behaves most similarly to F-tractin and Lifeact. However, it does not colocalise with mCherry-actin in dynamic ruffles, and may preferentially bind stable actin filaments. All 4 Affimers label F-actin in methanol fixed cells, while only Affimer 14 labels F-actin after paraformaldehyde fixation. eGFP-Affimer 6 has potential for use in selectively imaging the stable actin cytoskeleton in live cells, while all 4 Affimers are strong alternatives to phalloidin for labelling F-actin in fixed cells.

Umbilical cord mesenchymal stem cells labeled with multimodal iron oxide nanoparticles with fluorescent and magnetic properties: application for in vivo cell tracking

PubMed Central

Sibov, Tatiana T; Pavon, Lorena F; Miyaki, Liza A; Mamani, Javier B; Nucci, Leopoldo P; Alvarim, Larissa T; Silveira, Paulo H; Marti, Luciana C; Gamarra, LF

2014-01-01

Here we describe multimodal iron oxide nanoparticles conjugated to Rhodamine-B (MION-Rh), their stability in culture medium, and subsequent validation of an in vitro protocol to label mesenchymal stem cells from umbilical cord blood (UC-MSC) with MION-Rh. These cells showed robust labeling in vitro without impairment of their functional properties, the viability of which were evaluated by proliferation kinetic and ultrastructural analyzes. Thus, labeled cells were infused into striatum of adult male rats of animal model that mimic late onset of Parkinson’s disease and, after 15 days, it was observed that cells migrated along the medial forebrain bundle to the substantia nigra as hypointense spots in T2 magnetic resonance imaging. These data were supported by short-term magnetic resonance imaging. Studies were performed in vivo, which showed that about 5 × 105 cells could be efficiently detected in the short term following infusion. Our results indicate that these labeled cells can be efficiently tracked in a neurodegenerative disease model. PMID:24531365

Lgr6 labels a rare population of mammary gland progenitor cells that are able to originate luminal mammary tumours

PubMed Central

Messal, Hendrik A.; Andersson, Agneta B.; Ruiz, E. Josue; Gerling, Marco; Douagi, Iyadh; Spencer-Dene, Bradley; Musch, Alexandra; Mitter, Richard; Bhaw, Leena; Stone, Richard; Bornhorst, Dorothee; Sesay, Abdul K.; Jonkers, Jos; Stamp, Gordon; Malanchi, Ilaria; Toftgård, Rune; Behrens, Axel

2018-01-01

The mammary gland is composed of a complex cellular hierarchy with unusual postnatal plasticity. The identities of stem/progenitor cell populations, as well as tumour-initiating cells that give rise to breast cancer, are incompletely understood. Here we show that Lgr6 marks rare populations of cells in both basal and luminal mammary gland compartments in mice. Lineage tracing analysis showed that Lgr6+ cells are unipotent progenitors, which expand clonally during puberty but diminish in adulthood. In pregnancy or upon stimulation with ovarian hormones, adult Lgr6+ cells regained proliferative potency and their progeny formed alveoli over repeated pregnancies. Oncogenic mutations in Lgr6+ cells resulted in expansion of luminal cells, culminating in mammary gland tumours. Conversely, depletion of Lgr6+ cells in the MMTV-PyMT model of mammary tumourigenesis significantly impaired tumour growth. Thus, Lgr6 marks mammary gland progenitor cells that can initiate tumours, and cells of luminal breast tumours required for efficient tumour maintenance. PMID:27798604

Micro-Computed Tomography Detection of Gold Nanoparticle-Labelled Mesenchymal Stem Cells in the Rat Subretinal Layer.

PubMed

Mok, Pooi Ling; Leow, Sue Ngein; Koh, Avin Ee-Hwan; Mohd Nizam, Hairul Harun; Ding, Suet Lee Shirley; Luu, Chi; Ruhaslizan, Raduan; Wong, Hon Seng; Halim, Wan Haslina Wan Abdul; Ng, Min Hwei; Idrus, Ruszymah Binti Hj; Chowdhury, Shiplu Roy; Bastion, Catherine Mae-Lynn; Subbiah, Suresh Kumar; Higuchi, Akon; Alarfaj, Abdullah A; Then, Kong Yong

2017-02-08

Mesenchymal stem cells are widely used in many pre-clinical and clinical settings. Despite advances in molecular technology; the migration and homing activities of these cells in in vivo systems are not well understood. Labelling mesenchymal stem cells with gold nanoparticles has no cytotoxic effect and may offer suitable indications for stem cell tracking. Here, we report a simple protocol to label mesenchymal stem cells using 80 nm gold nanoparticles. Once the cells and particles were incubated together for 24 h, the labelled products were injected into the rat subretinal layer. Micro-computed tomography was then conducted on the 15th and 30th day post-injection to track the movement of these cells, as visualized by an area of hyperdensity from the coronal section images of the rat head. In addition, we confirmed the cellular uptake of the gold nanoparticles by the mesenchymal stem cells using transmission electron microscopy. As opposed to other methods, the current protocol provides a simple, less labour-intensive and more efficient labelling mechanism for real-time cell tracking. Finally, we discuss the potential manipulations of gold nanoparticles in stem cells for cell replacement and cancer therapy in ocular disorders or diseases.

Micro-Computed Tomography Detection of Gold Nanoparticle-Labelled Mesenchymal Stem Cells in the Rat Subretinal Layer

PubMed Central

Mok, Pooi Ling; Leow, Sue Ngein; Koh, Avin Ee-Hwan; Mohd Nizam, Hairul Harun; Ding, Suet Lee Shirley; Luu, Chi; Ruhaslizan, Raduan; Wong, Hon Seng; Halim, Wan Haslina Wan Abdul; Ng, Min Hwei; Idrus, Ruszymah Binti Hj.; Chowdhury, Shiplu Roy; Bastion, Catherine Mae-Lynn; Subbiah, Suresh Kumar; Higuchi, Akon; Alarfaj, Abdullah A.; Then, Kong Yong

2017-01-01

Mesenchymal stem cells are widely used in many pre-clinical and clinical settings. Despite advances in molecular technology; the migration and homing activities of these cells in in vivo systems are not well understood. Labelling mesenchymal stem cells with gold nanoparticles has no cytotoxic effect and may offer suitable indications for stem cell tracking. Here, we report a simple protocol to label mesenchymal stem cells using 80 nm gold nanoparticles. Once the cells and particles were incubated together for 24 h, the labelled products were injected into the rat subretinal layer. Micro-computed tomography was then conducted on the 15th and 30th day post-injection to track the movement of these cells, as visualized by an area of hyperdensity from the coronal section images of the rat head. In addition, we confirmed the cellular uptake of the gold nanoparticles by the mesenchymal stem cells using transmission electron microscopy. As opposed to other methods, the current protocol provides a simple, less labour-intensive and more efficient labelling mechanism for real-time cell tracking. Finally, we discuss the potential manipulations of gold nanoparticles in stem cells for cell replacement and cancer therapy in ocular disorders or diseases. PMID:28208719

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

PubMed

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

2016-04-04

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

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

NASA Astrophysics Data System (ADS)

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

2016-03-01

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

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

PubMed

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

2015-01-01

Diabetic skin wounds lack the ability to heal properly and constitute a major and significant complication of diabetes. Nontraumatic lower extremity amputations are the number one complication of diabetic skin wounds. The complexity of their pathophysiology requires an intervention at many levels to enhance healing and wound closure. Stem cells are a promising treatment for diabetic skin wounds as they have the ability to correct abnormal healing. Stem cell factor (SCF), a chemokine expressed in the skin, can induce stem cells migration, however the role of SCF in diabetic skin wound healing is still unknown. We hypothesize that SCF would correct the impairment and promote the healing of diabetic skin wounds. Our results show that SCF improved wound closure in diabetic mice and increased HIF-1α and vascular endothelial growth factor (VEGF) expression levels in these wounds. SCF treatment also enhanced the migration of red fluorescent protein (RFP)-labeled skin stem cells via in utero intra-amniotic injection of lenti-RFP at E8. Interestingly these RFP+ cells are present in the epidermis, stain negative for K15, and appear to be distinct from the already known hair follicle stem cells. These results demonstrate that SCF improves diabetic wound healing in part by increasing the recruitment of a unique stem cell population present in the skin. © 2015 by the Wound Healing Society.

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.

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

PubMed

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

2016-05-13

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.

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

PubMed Central

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

2010-01-01

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

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

PubMed

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

2014-01-01

Correlative fluorescence microscopy combined with scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, STEM can be accomplished in two ways. The microchip with the labeled cells and one microchip with a spacer are assembled into a special microfluidic device and imaged with dedicated high-voltage STEM. Alternatively, thin edges of cells can be studied with environmental scanning electron microscopy with a STEM detector, by placing a microchip with cells in a cooled wet environment. © 2014 Elsevier Inc. All rights reserved.

Fluorescent labeling of tetracysteine-tagged proteins in intact cells.

PubMed

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

2010-09-01

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.

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

A novel label-free cell-based assay technology using biolayer interferometry.

PubMed

Verzijl, D; Riedl, T; Parren, P W H I; Gerritsen, A F

2017-01-15

Biolayer interferometry (BLI) is a well-established optical label-free technique to study biomolecular interactions. Here we describe for the first time a cell-based BLI (cBLI) application that allows label-free real-time monitoring of signal transduction in living cells. Human A431 epidermoid carcinoma cells were captured onto collagen-coated biosensors and serum-starved, followed by exposure to agonistic compounds targeting various receptors, while recording the cBLI signal. Stimulation of the epidermal growth factor receptor (EGFR) with EGF, the β 2 -adrenoceptor with dopamine, or the hepatocyte growth factor receptor (HGFR/c-MET) with an agonistic antibody resulted in distinct cBLI signal patterns. We show that the mechanism underlying the observed changes in cBLI signal is mediated by rearrangement of the actin cytoskeleton, a process referred to as dynamic mass redistribution (DMR). A panel of ligand-binding blocking and non-blocking anti-EGFR antibodies was used to demonstrate that this novel BLI application can be efficiently used as a label-free cellular assay for compound screening and characterization. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Multi-Isotope Secondary Ion Mass Spectrometry Combining Heavy Water 2H with 15N Labeling As Complementary Tracers for Metabolic Heterogeneity at the Single-Cell Level

NASA Astrophysics Data System (ADS)

Kopf, S.; McGlynn, S.; Cowley, E.; Green, A.; Newman, D. K.; Orphan, V. J.

2014-12-01

Metabolic rates of microbial communities constitute a key physiological parameter for understanding the in situ growth constraints for life in any environment. Isotope labeling techniques provide a powerful approach for measuring such biological activity, due to the use of isotopically enriched substrate tracers whose incorporation into biological materials can be detected with high sensitivity by isotope-ratio mass spectrometry. Nano-meter scale secondary ion mass spectrometry (NanoSIMS) combined with stable isotope labeling provides a unique tool for studying the spatiometabolic activity of microbial populations at the single cell level in order to assess both community structure and population diversity. However, assessing the distribution and range of microbial activity in complex environmental systems with slow-growing organisms, diverse carbon and nitrogen sources, or heterotrophic subpopulations poses a tremendous technical challenge because the introduction of isotopically labeled substrates frequently changes the nutrient availability and can inflate or bias measures of activity. Here, we present the use of hydrogen isotope labeling with deuterated water as an important new addition to the isotopic toolkit and apply it for the determination of single cell microbial activities by NanoSIMS imaging. This tool provides a labeling technique that minimally alters any aquatic chemical environment, can be administered with strong labels even in minimal addition (natural background is very low), is an equally universal substrate for all forms of life even in complex, carbon and nitrogen saturated systems, and can be combined with other isotopic tracers. The combination of heavy water labeling with the most commonly used NanoSIMS tracer, 15N, is technically challenging but opens up a powerful new set of multi-tracer experiments for the study of microbial activity in complex communities. We present the first truly simultaneous single cell triple isotope system

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

PubMed Central

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

2010-01-01

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. PMID:20368463

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

DOE PAGES

Nelson, Johanna; Huang, Xiaojing; Steinbrener, Jan; ...

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

The retina of the shovel-nosed ray, Rhinobatos batillum (Rhinobatidae): morphology and quantitative analysis of the ganglion, amacrine and bipolar cell populations.

PubMed

Collin, S P

1988-01-01

A light microscopy study of the retina of the shovel-nosed ray, Rhinobatos batillum (Rhinobatidae) has revealed a duplex retina with a rod to cone ratio between 4:1 and 6:1. The inner nuclear layer consists of three layers of large horizontal cells, tightly packed, stellate bipolar cells, and up to three substrata of amacrine cells. The collaterals of the many supporting Müller cells project from the inner to the outer limiting membrane and divide the retina into many subunits. The cells of the ganglion cell layer are distributed into two layers, although a large proportion of ganglion cells are also displaced into the inner plexiform and inner nuclear layers. Topographic analysis of the cells in the ganglion cell layer, inner plexiform and inner nuclear layers reveals a number of regional specializations or "areae centrales". Ganglion cells were retrogradely-labelled with cobalt-lysine from the optic nerve, and three sub-populations of neurons characterized on their soma size and position. Small (20-50 microns2), large (80-300 microns2) and giant (greater than 300 microns2) sub-populations of ganglion cells each revealed distinct retinal specializations with peak densities of 3 x 10(3), 1.25 x 10(3) and 1.57 x 10(3) cells per mm2, respectively. Topographical comparison between Nissl-stained and retrogradely-labelled ganglion cell populations have established that a maximum of 20% in the "area centralis", and 75% in unspecialized, peripheral regions of the retina are non-ganglion cells. Out of a total of 210,566 cells in the ganglion cell layer, 49% were found to be non-ganglion cells. Iso-density contour maps of amacrine and bipolar cell distributions also reveal some specializations. These cell concentrations lie in corresponding regions to areas of increased density in the large and giant ganglion cell populations, suggesting some functional association.

Dual-modality wide-field photothermal quantitative phase microscopy and depletion of cell populations

NASA Astrophysics Data System (ADS)

Turko, Nir A.; Barnea, Itay; Blum, Omry; Korenstein, Rafi; Shaked, Natan T.

2015-03-01

We review our dual-modality technique for quantitative imaging and selective depletion of populations of cells based on wide-field photothermal (PT) quantitative phase imaging and simultaneous PT cell extermination. The cells are first labeled by plasmonic gold nanoparticles, which evoke local plasmonic resonance when illuminated by light in a wavelength corresponding to their specific plasmonic resonance peak. This reaction creates changes of temperature, resulting in changes of phase. This phase changes are recorded by a quantitative phase microscope (QPM), producing specific imaging contrast, and enabling bio-labeling in phase microscopy. Using this technique, we have shown discrimination of EGFR over-expressing (EGFR+) cancer cells from EGFR under-expressing (EGFR-) cancer cells. Then, we have increased the excitation power in order to evoke greater temperatures, which caused specific cell death, all under real-time phase acquisition using QPM. Close to 100% of all EGFR+ cells were immediately exterminated when illuminated with the strong excitation beam, while all EGFR- cells survived. For the second experiment, in order to simulate a condition where circulating tumor cells (CTCs) are present in blood, we have mixed the EGFR+ cancer cells with white blood cells (WBCs) from a healthy donor. Here too, we have used QPM to observe and record the phase of the cells as they were excited for selective visualization and then exterminated. The WBCs survival rate was over 95%, while the EGFR+ survival rate was under 5%. The technique may be the basis for real-time detection and controlled treatment of CTCs.

Photoaffinity-labeling and fluorescence-distribution studies of gonadotropin-releasing hormone receptors in ovarian granulosa cells.

PubMed Central

Hazum, E; Nimrod, A

1982-01-01

Photoaffinity labeling of rat ovarian granulosa cells and membrane preparations with a bioactive photoaffinity derivative of gonadotropin-releasing hormone resulted in identification of two specific components with apparent molecular weights of 60,000 and 54,000. Fluorescent visualization of gonadotropin-releasing hormone receptors in these cells, by using a bioactive rhodamine derivative of the hormone, indicated that the fluorescently labeled receptors were initially distributed uniformly on the cell surface and then formed patches that subsequently internalized (at 37 degrees C) into endocytic vesicles. These processes were dependent on specific binding sites for the rhodamine-labeled peptide on the granulosa cells. These studies may provide an experimental basis for understanding the molecular events involved in the action of the hormone in the ovary. Images PMID:6281784

Radionuclide and Fluorescence Imaging of Clear Cell Renal Cell Carcinoma Using Dual Labeled Anti-Carbonic Anhydrase IX Antibody G250.

PubMed

Muselaers, Constantijn H J; Rijpkema, Mark; Bos, Desirée L; Langenhuijsen, Johan F; Oyen, Wim J G; Mulders, Peter F A; Oosterwijk, Egbert; Boerman, Otto C

2015-08-01

Tumor targeted optical imaging using antibodies labeled with near infrared fluorophores is a sensitive imaging modality that might be used during surgery to assure complete removal of malignant tissue. We evaluated the feasibility of dual modality imaging and image guided surgery with the dual labeled anti-carbonic anhydrase IX antibody preparation (111)In-DTPA-G250-IRDye800CW in mice with intraperitoneal clear cell renal cell carcinoma. BALB/c nu/nu mice with intraperitoneal SK-RC-52 lesions received 10 μg DTPA-G250-IRDye800CW labeled with 15 MBq (111)In or 10 μg of the dual labeled irrelevant control antibody NUH-82 (20 mice each). To evaluate when tumors could be detected, 4 mice per group were imaged weekly during 5 weeks with single photon emission computerized tomography/computerized tomography and the fluorescence imaging followed by ex vivo biodistribution studies. As early as 1 week after tumor cell inoculation single photon emission computerized tomography and fluorescence images showed clear delineation of intraperitoneal clear cell renal cell carcinoma with good concordance between single photon emission computerized tomography/computerized tomography and fluorescence images. The high and specific accumulation of the dual labeled antibody conjugate in tumors was confirmed in the biodistribution studies. Maximum tumor uptake was observed 1 week after inoculation (mean ± SD 58.5% ± 18.7% vs 5.6% ± 2.3% injected dose per gm for DTPA-G250-IRDye800CW vs NUH-82, respectively). High tumor uptake was also observed at other time points. This study demonstrates the feasibility of dual modality imaging with dual labeled antibody (111)In-DTPA-G250-IRDye800CW in a clear cell renal cell carcinoma model. Results indicate that preoperative and intraoperative detection of carbonic anhydrase IX expressing tumors, positive resection margins and metastasis might be feasible with this approach. Copyright © 2015 American Urological Association Education and Research

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

PubMed

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

2011-11-01

Microflow cytometry represents a promising tool for the investigation of diagnostic and prognostic cellular cancer markers, particularly if integrated within a device that allows primary cells to be freshly isolated from the solid tumour biopsies that more accurately reflect patient-specific in vivo tissue microenvironments at the time of staining. However, current tissue processing techniques involve several sequential stages with concomitant cell losses, and as such are inappropriate for use with small biopsies. Accordingly, we present a simple method for combined antibody-labelling and dissociation of heterogeneous cells from a tumour mass, which reduces the number of processing steps. Perfusion of ex vivo tissue at 4°C with antibodies and enzymes slows cellular activity while allowing sufficient time for the diffusion of minimally active enzymes. In situ antibody-labelled cells are then dissociated at 37°C from the tumour mass, whereupon hydrogel-filled channels allow the release of relatively low cell numbers (<1000) into a biomimetic microenvironment. This novel approach to sample processing is then further integrated with hydrogel-based electrokinetic transport of the freshly liberated fluorescent cells for downstream detection. It is anticipated that this integrated microfluidic methodology will have wide-ranging biomedical and clinical applications. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cell sorting apparatus

NASA Technical Reports Server (NTRS)

Yen, Shiao-Ping S. (Inventor); Rembaum, Alan (Inventor); Molday, Robert S. (Inventor)

1980-01-01

Polymeric functional microspheres containing metal or metal compounds are formed by addition polymerization of a covalently bondable olefinic monomer such as hydroxyethylmethacrylate in the presence of finely divided metal or metal oxide particles, such as iron, gold, platinum or magnetite, which are embedded in the resulting microspheres. The microspheres can be covalently bonded to chemotherapeutic agents, antibodies, or other proteins providing a means for labeling or separating labeled cells. Labeled cells or microspheres can be concentrated at a specific body location such as in the vicinity of a malignant tumor by applying a magnetic field to the location and then introducing the magnetically attractable microspheres or cells into the circulatory system of the subject. Labeled cells can be separated from a cell mixture by applying a predetermined magnetic field to a tube in which the mixture is flowing. After collection of the labeled cells, the magnetic field is discontinued and the labeled sub-cell population recovered.

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

101 Labeled Brain Images and a Consistent Human Cortical Labeling Protocol

PubMed Central

Klein, Arno; Tourville, Jason

2012-01-01

We introduce the Mindboggle-101 dataset, the largest and most complete set of free, publicly accessible, manually labeled human brain images. To manually label the macroscopic anatomy in magnetic resonance images of 101 healthy participants, we created a new cortical labeling protocol that relies on robust anatomical landmarks and minimal manual edits after initialization with automated labels. The “Desikan–Killiany–Tourville” (DKT) protocol is intended to improve the ease, consistency, and accuracy of labeling human cortical areas. Given how difficult it is to label brains, the Mindboggle-101 dataset is intended to serve as brain atlases for use in labeling other brains, as a normative dataset to establish morphometric variation in a healthy population for comparison against clinical populations, and contribute to the development, training, testing, and evaluation of automated registration and labeling algorithms. To this end, we also introduce benchmarks for the evaluation of such algorithms by comparing our manual labels with labels automatically generated by probabilistic and multi-atlas registration-based approaches. All data and related software and updated information are available on the http://mindboggle.info/data website. PMID:23227001

Label-free monitoring of cell death induced by oxidative stress in living human cells using terahertz ATR spectroscopy

PubMed Central

Zou, Yi; Liu, Qiao; Yang, Xia; Huang, Hua-Chuan; Li, Jiang; Du, Liang-Hui; Li, Ze-Ren; Zhao, Jian-Heng; Zhu, Li-Guo

2017-01-01

We demonstrated that attenuated total reflectance terahertz time-domain spectroscopy (ATR THz-TDS) is able to monitor oxidative stress response of living human cells, which is proven in this work that it is an efficient non-invasive, label-free, real-time and in situ monitoring of cell death. Furthermore, the dielectric constant and dielectric loss of cultured living human breast epithelial cells, and along with their evolution under oxidative stress response induced by high concentration of H2O2, were quantitatively determined in the work. Our observation and results were finally confirmed using standard fluorescence-labeled flow cytometry measurements and visible fluorescence imaging. PMID:29359084

Negro, Black, Black African, African Caribbean, African American or what? Labelling African origin populations in the health arena in the 21st century

PubMed Central

Agyemang, C.; Bhopal, R.; Bruijnzeels, M.

2005-01-01

Broad terms such as Black, African, or Black African are entrenched in scientific writings although there is considerable diversity within African descent populations and such terms may be both offensive and inaccurate. This paper outlines the heterogeneity within African populations, and discusses the strengths and limitations of the term Black and related labels from epidemiological and public health perspectives in Europe and the USA. This paper calls for debate on appropriate terminologies for African descent populations and concludes with the proposals that (1) describing the population under consideration is of paramount importance (2) the word African origin or simply African is an appropriate and necessary prefix for an ethnic label, for example, African Caribbean or African Kenyan or African Surinamese (3) documents should define the ethnic labels (4) the label Black should be phased out except when used in political contexts. PMID:16286485

Labelling and targeted ablation of specific bipolar cell types in the zebrafish retina

PubMed Central

2009-01-01

Background Development of a functional retina depends on regulated differentiation of several types of neurons and generation of a highly complex network between the different types of neurons. In addition, each type of retinal neuron includes several distinct morphological types. Very little is known about the mechanisms responsible for generating this diversity of retinal neurons, which may also display specific patterns of regional distribution. Results In a screen in zebrafish, using a trapping vector carrying an engineered yeast Gal4 transcription activator and a UAS:eGFP reporter cassette, we have identified two transgenic lines of zebrafish co-expressing eGFP and Gal4 in specific subsets of retinal bipolar cells. The eGFP-labelling facilitated analysis of axon terminals within the inner plexiform layer of the adult retina and showed that the fluorescent bipolar cells correspond to previously defined morphological types. Strong regional restriction of eGFP-positive bipolar cells to the central part of the retina surrounding the optic nerve was observed in adult zebrafish. Furthermore, we achieved specific ablation of the labelled bipolar cells in 5 days old larvae, using a bacterial nitroreductase gene under Gal4-UAS control in combination with the prodrug metronidazole. Following prodrug treatment, nitroreductase expressing bipolar cells were efficiently ablated without affecting surrounding retina architecture, and recovery occurred within a few days due to increased generation of new bipolar cells. Conclusion This report shows that enhancer trapping can be applied to label distinct morphological types of bipolar cells in the zebrafish retina. The genetic labelling of these cells yielded co-expression of a modified Gal4 transcription activator and the fluorescent marker eGFP. Our work also demonstrates the potential utility of the Gal4-UAS system for induction of other transgenes, including a bacterial nitroreductase fusion gene, which can facilitate

Characterization, in vitro cytotoxicity assessment, and in vivo visualization of multimodal, RITC-labeled, silica-coated magnetic nanoparticles for labeling human cord blood-derived mesenchymal stem cells.

PubMed

Park, Ki-Soo; Tae, Jinsung; Choi, Bongkum; Kim, Young-Seok; Moon, Cheol; Kim, Sa-Hyun; Lee, Han-Sin; Kim, Jinhyun; Kim, Junsung; Park, Jaeberm; Lee, Jung-Hee; Lee, Jong Eun; Joh, Jae-Won; Kim, Sungjoo

2010-04-01

Live imaging is a powerful technique that can be used to characterize the fate and location of stem cells in animal models. Here we investigated the characteristics and in vitro cytotoxicity of human mesenchymal stem cells (MSCs) labeled with silica-coated magnetic nanoparticles incorporating rhodamine B isothiocyanate, MNPs@SiO2(RITC). We also conducted various in vivo-uptake tests with nanoparticle-labeled human MSCs. MNPs@SiO2(RITC) showed photostability against ultraviolet light exposure and were nontoxic to human MSCs, based on the MTT, apoptosis, and cell cycle arrest assays. In addition, MNPs@SiO2(RITC) did not affect the surface phenotype or morphology of human MSCs. We also demonstrated that MNPs@SiO2(RITC) have stable retention properties in MSCs in vitro. Furthermore, using optical and magnetic resonance imaging, we successfully detected a visible signal from labeled human MSCs that were transplanted into NOD.CB17-Prkdc(SCID) (NOD-SCID) mice. These results demonstrate that MNPs@SiO2(RITC) are biocompatible and useful tools for human MSC labeling and bioimaging. The characteristics and in vitro cytotoxicity of human mesenchymal stem cells (MSCs) labeled with silica-coated magnetic nanoparticles incorporating rhodamine B isothiocyanate, RITC were investigated in this study. RITC showed photostability against ultraviolet light exposure and was nontoxic to human MSCs. Using both optical and magnetic resonance imaging, successful detection of signal from labeled human MSCs transplanted into mice is demonstrated. Copyright 2010 Elsevier Inc. All rights reserved.

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

Micrometer-sized iron oxide particle labeling of mesenchymal stem cells for magnetic resonance imaging-based monitoring of cartilage tissue engineering.

PubMed

Saldanha, Karl J; Doan, Ryan P; Ainslie, Kristy M; Desai, Tejal A; Majumdar, Sharmila

2011-01-01

To examine mesenchymal stem cell (MSC) labeling with micrometer-sized iron oxide particles (MPIOs) for magnetic resonance imaging (MRI)-based tracking and its application to monitoring articular cartilage regeneration. Rabbit MSCs were labeled using commercial MPIOs. In vitro MRI was performed with gradient echo (GRE) and spin echo (SE) sequences at 3T and quantitatively characterized using line profile and region of interest analysis. Ex vivo MRI of hydrogel-encapsulated labeled MSCs implanted within a bovine knee was performed with spoiled GRE (SPGR) and T(1ρ) sequences. Fluorescence microscopy, labeling efficiency, and chondrogenesis of MPIO-labeled cells were also examined. MPIO labeling results in efficient contrast uptake and signal loss that can be visualized and quantitatively characterized via MRI. SPGR imaging of implanted cells results in ex vivo detection within native tissue, and T(1ρ) imaging is unaffected by the presence of labeled cells immediately following implantation. MPIO labeling does not affect quantitative glycosaminoglycan production during chondrogenesis, but iron aggregation hinders extracellular matrix visualization. This aggregation may result from excess unincorporated particles following labeling and is an issue that necessitates further investigation. This study demonstrates the promise of MPIO labeling for monitoring cartilage regeneration and highlights its potential in the development of cell-based tissue engineering strategies. Published by Elsevier Inc.

Magnetic Resonance Imaging of Ferumoxytol-Labeled Human Mesenchymal Stem Cells in the Mouse Brain.

PubMed

Lee, Na Kyung; Kim, Hyeong Seop; Yoo, Dongkyeom; Hwang, Jung Won; Choi, Soo Jin; Oh, Wonil; Chang, Jong Wook; Na, Duk L

2017-02-01

The success of stem cell therapy is highly dependent on accurate delivery of stem cells to the target site of interest. Possible ways to track the distribution of MSCs in vivo include the use of reporter genes or nanoparticles. The U.S. Food and Drug Administration (FDA) has approved ferumoxytol (Feraheme® [USA], Rienso® [UK]) as a treatment for iron deficiency anemia. Ferumoxytol is an ultrasmall superparamagnetic iron oxide nanoparticle (USPIO) that has recently been used to track the fate of transplanted cells using magnetic resonance imaging (MRI). The major objectives of this study were to demonstrate the feasibility of labeling hUCB-MSCs with ferumoxytol and to observe, through MRI, the engraftment of ferumoxytol-labeled human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) delivered via stereotactic injection into the hippocampi of a transgenic mouse model of familial Alzheimer's disease (5XFAD). Ferumoxytol had no toxic effects on the viability or stemness of hUCB-MSCs when assessed in vitro. Through MRI, hypointense signals were discernible at the site where ferumoxytol-labeled human MSCs were injected. Iron-positive areas were also observed in the engrafted hippocampi. The results from this study support the use of nanoparticle labeling to monitor transplanted MSCs in real time as a follow-up for AD stem cell therapy in the clinical field.

Fluorescently labeled dengue viruses as probes to identify antigen-specific memory B cells by multiparametric flow cytometry.

PubMed

Woda, Marcia; Mathew, Anuja

2015-01-01

Low frequencies of memory B cells in the peripheral blood make it challenging to measure the functional and phenotypic characteristics of this antigen experienced subset of B cells without in vitro culture. To date, reagents are lacking to measure ex vivo frequencies of dengue virus (DENV)-specific memory B cells. We wanted to explore the possibility of using fluorescently labeled DENV as probes to detect antigen-specific memory B cells in the peripheral blood of DENV immune individuals. Alexa Fluor dye-labeled DENV yielded viable virus that could be stored at -80°C for long periods of time. Using a careful gating strategy and methods to decrease non-specific binding, we were able to identify a small frequency of B cells from dengue immune individuals that bound labeled DENV. Sorted DENV(+) B cells from immune, but not naïve donors secreted antibodies that bound DENV after in vitro stimulation. Overall, Alexa Fluor dye-labeled DENVs are useful reagents to enable the detection and characterization of memory B cells in DENV immune individuals. Copyright © 2014 Elsevier B.V. All rights reserved.

Fluorescently labeled dengue viruses as probes to identify antigen-specific memory B cells by multiparametric flow cytometry

PubMed Central

Woda, Marcia; Mathew, Anuja

2015-01-01

Low frequencies of memory B cells in the peripheral blood make it challenging to measure the functional and phenotypic characteristics of this antigen experienced subset of B cells without in vitro culture. To date, reagents are lacking to measure ex vivo frequencies of dengue virus (DENV)-specific memory B cells. We wanted to explore the possibility of using fluorescently labeled DENV as probes to detect antigen-specific memory B cells in the peripheral blood of DENV immune individuals. Alexa Fluor dye-labeled DENV yielded viable virus that could be stored at −80°C for long periods of time. Using a careful gating strategy and methods to decrease non-specific binding, we were able to identify a small frequency of B cells from dengue immune individuals that bound labeled DENV. Sorted DENV+ B cells from immune, but not naïve donors secreted antibodies that bound intact virions after in vitro stimulation. Overall, Alexa Fluor dye labeled -DENV are useful reagents to enable the detection and characterization of memory B cells in DENV immune individuals. PMID:25497702

Isotope labeling of proteins in insect cells.

PubMed

Skora, Lukasz; Shrestha, Binesh; Gossert, Alvar D

2015-01-01

Protein targets of contemporary research are often membrane proteins, multiprotein complexes, secreted proteins, or other proteins of human origin. These are difficult to express in the standard expression host used for most nuclear magnetic resonance (NMR) studies, Escherichia coli. Insect cells represent an attractive alternative, since they have become a well-established expression system and simple solutions have been developed for generation of viruses to efficiently introduce the target protein DNA into cells. Insect cells enable production of a larger fraction of the human proteome in a properly folded way than bacteria, as insect cells have a very similar set of cytosolic chaperones and a closely related secretory pathway. Here, the limited and defined glycosylation pattern that insect cells produce is an advantage for structural biology studies. For these reasons, insect cells have been established as the most widely used eukaryotic expression host for crystallographic studies. In the past decade, significant advancements have enabled amino acid type-specific as well as uniform isotope labeling of proteins in insect cells, turning them into an attractive expression host for NMR studies. © 2015 Elsevier Inc. All rights reserved.

Magnetic Resonance Imaging Tracking of Ferumoxytol-Labeled Human Neural Stem Cells: Studies Leading to Clinical Use

PubMed Central

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

2013-01-01

Numerous stem cell-based therapies are currently under clinical investigation, including the use of neural stem cells (NSCs) as delivery vehicles to target therapeutic agents to invasive brain tumors. The ability to monitor the time course, migration, and distribution of stem cells following transplantation into patients would provide critical information for optimizing treatment regimens. No effective cell-tracking methodology has yet garnered clinical acceptance. A highly promising noninvasive method for monitoring NSCs and potentially other cell types in vivo involves preloading them with ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) to enable cell tracking using magnetic resonance imaging (MRI). We report here the preclinical studies that led to U.S. Food and Drug Administration approval for first-in-human investigational use of ferumoxytol to label NSCs prior to transplantation into brain tumor patients, followed by surveillance serial MRI. A combination of heparin, protamine sulfate, and ferumoxytol (HPF) was used to label the NSCs. HPF labeling did not affect cell viability, growth kinetics, or tumor tropism in vitro, and it enabled MRI visualization of NSC distribution within orthotopic glioma xenografts. MRI revealed dynamic in vivo NSC distribution at multiple time points following intracerebral or intravenous injection into glioma-bearing mice that correlated with histological analysis. Preclinical safety/toxicity studies of intracerebrally administered HPF-labeled NSCs in mice were also performed, and they showed no significant clinical or behavioral changes, no neuronal or systemic toxicities, and no abnormal accumulation of iron in the liver or spleen. These studies support the clinical use of ferumoxytol labeling of cells for post-transplant MRI visualization and tracking. PMID:24014682

Skeletal stem cell isolation: A review on the state-of-the-art microfluidic label-free sorting techniques.

PubMed

Xavier, Miguel; Oreffo, Richard O C; Morgan, Hywel

2016-01-01

Skeletal stem cells (SSC) are a sub-population of bone marrow stromal cells that reside in postnatal bone marrow with osteogenic, chondrogenic and adipogenic differentiation potential. SSCs reside only in the bone marrow and have organisational and regulatory functions in the bone marrow microenvironment and give rise to the haematopoiesis-supportive stroma. Their differentiation capacity is restricted to skeletal lineages and therefore the term SSC should be clearly distinguished from mesenchymal stem cells which are reported to exist in extra-skeletal tissues and, critically, do not contribute to skeletal development. SSCs are responsible for the unique regeneration capacity of bone and offer unlimited potential for application in bone regenerative therapies. A current unmet challenge is the isolation of homogeneous populations of SSCs, in vitro, with homogeneous regeneration and differentiation capacities. Challenges that limit SSC isolation include a) the scarcity of SSCs in bone marrow aspirates, estimated at between 1 in 10-100,000 mononuclear cells; b) the absence of specific markers and thus the phenotypic ambiguity of the SSC and c) the complexity of bone marrow tissue. Microfluidics provides innovative approaches for cell separation based on bio-physical features of single cells. Here we review the physical principles underlying label-free microfluidic sorting techniques and review their capacity for stem cell selection/sorting from complex (heterogeneous) samples. Copyright © 2016 Elsevier Inc. All rights reserved.

Glioblastoma cells labeled by robust Raman tags for enhancing imaging contrast.

PubMed

Huang, Li-Ching; Chang, Yung-Ching; Wu, Yi-Syuan; Sun, Wei-Lun; Liu, Chan-Chuan; Sze, Chun-I; Chen, Shiuan-Yeh

2018-05-01

Complete removal of a glioblastoma multiforme (GBM), a highly malignant brain tumor, is challenging due to its infiltrative characteristics. Therefore, utilizing imaging agents such as fluorophores to increase the contrast between GBM and normal cells can help neurosurgeons to locate residual cancer cells during image guided surgery. In this work, Raman tag based labeling and imaging for GBM cells in vitro is described and evaluated. The cell membrane of a GBM adsorbs a substantial amount of functionalized Raman tags through overexpression of the epidermal growth factor receptor (EGFR) and "broadcasts" stronger pre-defined Raman signals than normal cells. The average ratio between Raman signals from a GBM cell and autofluorescence from a normal cell can be up to 15. In addition, the intensity of these images is stable under laser illuminations without suffering from the severe photo-bleaching that usually occurs in fluorescent imaging. Our results show that labeling and imaging GBM cells via robust Raman tags is a viable alternative method to distinguish them from normal cells. This Raman tag based method can be used solely or integrated into an existing fluorescence system to improve the identification of infiltrative glial tumor cells around the boundary, which will further reduce GBM recurrence. In addition, it can also be applied/extended to other types of cancer to improve the effectiveness of image guided surgery.

Glioblastoma cells labeled by robust Raman tags for enhancing imaging contrast

PubMed Central

Huang, Li-Ching; Chang, Yung-Ching; Wu, Yi-Syuan; Sun, Wei-Lun; Liu, Chan-Chuan; Sze, Chun-I; Chen, Shiuan-Yeh

2018-01-01

Complete removal of a glioblastoma multiforme (GBM), a highly malignant brain tumor, is challenging due to its infiltrative characteristics. Therefore, utilizing imaging agents such as fluorophores to increase the contrast between GBM and normal cells can help neurosurgeons to locate residual cancer cells during image guided surgery. In this work, Raman tag based labeling and imaging for GBM cells in vitro is described and evaluated. The cell membrane of a GBM adsorbs a substantial amount of functionalized Raman tags through overexpression of the epidermal growth factor receptor (EGFR) and “broadcasts” stronger pre-defined Raman signals than normal cells. The average ratio between Raman signals from a GBM cell and autofluorescence from a normal cell can be up to 15. In addition, the intensity of these images is stable under laser illuminations without suffering from the severe photo-bleaching that usually occurs in fluorescent imaging. Our results show that labeling and imaging GBM cells via robust Raman tags is a viable alternative method to distinguish them from normal cells. This Raman tag based method can be used solely or integrated into an existing fluorescence system to improve the identification of infiltrative glial tumor cells around the boundary, which will further reduce GBM recurrence. In addition, it can also be applied/extended to other types of cancer to improve the effectiveness of image guided surgery. PMID:29760976

A Population of Progenitor Cells in the Basal and Intermediate Layers of the Murine Bladder Urothelium Contributes to Urothelial Development and Regeneration

PubMed Central

Colopy, Sara A.; Bjorling, Dale E.; Mulligan, William A.; Bushman, Wade

2014-01-01

Background Homeostatic maintenance and repair of the bladder urothelium has been attributed to proliferation of keratin 5-expressing basal cells (K5-BC) with subsequent differentiation into superficial cells. Recent evidence, however, suggests that the intermediate cell layer harbors a population of progenitor cells. We use label-retaining cell (LRC) methodology in conjunction with a clinically relevant model of uropathogenic Escherichia coli (UPEC)-induced injury to characterize urothelial ontogeny during development and in response to diffuse urothelial injury. Results In the developing urothelium, proliferating cells were dispersed throughout the K5-BC and intermediate cells layers, becoming progressively concentrated in the K5-BC layer with age. When 5-bromo-2-deoxyuridine (BrdU) was administered during urothelial development, LRCs in the adult were found within the K5-BC, intermediate, and superficial cell layers, the location dependent upon time of labeling. UPEC inoculation resulted in loss of the superficial cell layer followed by robust proliferation of K5-BCs and intermediate cells. LRCs within the K5-BC and intermediate cell layers proliferated in response to injury. Conclusions Urothelial development and regeneration following injury relies on proliferation of K5-BC and intermediate cells. The existence and proliferation of LRCs within both the K5-BC and intermediate cell layers suggests the presence of two populations of urothelial progenitor cells. PMID:24796293

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. Copyright © 2015 Elsevier Inc. All rights reserved.

Human neural progenitor cells retain viability, phenotype, proliferation, and lineage differentiation when labeled with a novel iron oxide nanoparticle, Molday ION Rhodamine B

PubMed Central

Shen, Wei-Bin; Plachez, Celine; Chan, Amanda; Yarnell, Deborah; Puche, Adam C; Fishman, Paul S; Yarowsky, Paul

2013-01-01

Ultrasmall superparamagnetic iron-oxide particles (USPIOs) loaded into stem cells have been suggested as a way to track stem cell transplantation with magnetic resonance imaging, but the labeling, and post-labeling proliferation, viability, differentiation, and retention of USPIOs within the stem cells have yet to be determined for each type of stem cell and for each type of USPIO. Molday ION Rhodamine B™ (BioPAL, Worcester, MA, USA) (MIRB) has been shown to be a USPIO labeling agent for mesenchymal stem cells, glial progenitor cells, and stem cell lines. In this study, we have evaluated MIRB labeling in human neuroprogenitor cells and found that human neuroprogenitor cells are effectively labeled with MIRB without use of transfection reagents. Viability, proliferation, and differentiation properties are unchanged between MIRB-labeled neuroprogenitors cells and unlabeled cells. Moreover, MIRB-labeled human neuroprogenitor cells can be frozen, thawed, and replated without loss of MIRB or even without loss of their intrinsic biology. Overall, those results show that MIRB has advantageous properties that can be used for cell-based therapy. PMID:24348036

Growth fraction in non-small cell lung cancer estimated by proliferating cell nuclear antigen and comparison with Ki-67 labeling and DNA flow cytometry data.

PubMed Central

Fontanini, G.; Pingitore, R.; Bigini, D.; Vignati, S.; Pepe, S.; Ruggiero, A.; Macchiarini, P.

1992-01-01

Results generated by the immunohistochemical staining with PC10, a new monoclonal antibody recognizing PCNA (a nuclear protein associated with cell proliferation) in formalin-fixed and paraffin-embedded tissue were compared with those of Ki-67 labeling and DNA flow cytometry in 47 consecutive non-small cell lung cancer (NSCLC). PCNA reactivity was observed in all samples and confined to the nuclei of cancer cells. Its frequency ranged from 0 to 80% (37.7 +/- 23.6) and larger sized, early-staged and DNA aneuploid tumors expressed a significant higher number of PCNA-reactive cells. The PCNA and Ki-67 labeling rates were closely correlated (r = 0.383, P = 0.009). By flow cytometry, we observed a good correlation among PCNA labeling and S-phase fraction (r = 0.422, P = .0093) and G1 phase (r = 0.303, P = .051) of the cell cycle. Results indicate that PCNA labeling with PC10 is a simple method for assessing the proliferative activity in formalin-fixed, paraffin-embedded tissue of NSCLC and correlates well with Ki-67 labeling and S-phase fraction of the cell cycle. Images Figure 2 PMID:1361306

Label-Free Optofluidic Nanobiosensor Enables Real-Time Analysis of Single-Cell Cytokine Secretion.

PubMed

Li, Xiaokang; Soler, Maria; Szydzik, Crispin; Khoshmanesh, Khashayar; Schmidt, Julien; Coukos, George; Mitchell, Arnan; Altug, Hatice

2018-06-01

Single-cell analysis of cytokine secretion is essential to understand the heterogeneity of cellular functionalities and develop novel therapies for multiple diseases. Unraveling the dynamic secretion process at single-cell resolution reveals the real-time functional status of individual cells. Fluorescent and colorimetric-based methodologies require tedious molecular labeling that brings inevitable interferences with cell integrity and compromises the temporal resolution. An innovative label-free optofluidic nanoplasmonic biosensor is introduced for single-cell analysis in real time. The nanobiosensor incorporates a novel design of a multifunctional microfluidic system with small volume microchamber and regulation channels for reliable monitoring of cytokine secretion from individual cells for hours. Different interleukin-2 secretion profiles are detected and distinguished from single lymphoma cells. The sensor configuration combined with optical spectroscopic imaging further allows us to determine the spatial single-cell secretion fingerprints in real time. This new biosensor system is anticipated to be a powerful tool to characterize single-cell signaling for basic and clinical research. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Retention of Ag-specific memory CD4+ T cells in the draining lymph node indicates lymphoid tissue resident memory populations.

PubMed

Marriott, Clare L; Dutton, Emma E; Tomura, Michio; Withers, David R

2017-05-01

Several different memory T-cell populations have now been described based upon surface receptor expression and migratory capabilities. Here we have assessed murine endogenous memory CD4 + T cells generated within a draining lymph node and their subsequent migration to other secondary lymphoid tissues. Having established a model response targeting a specific peripheral lymph node, we temporally labelled all the cells within draining lymph node using photoconversion. Tracking of photoconverted and non-photoconverted Ag-specific CD4 + T cells revealed the rapid establishment of a circulating memory population in all lymph nodes within days of immunisation. Strikingly, a resident memory CD4 + T cell population became established in the draining lymph node and persisted for several months in the absence of detectable migration to other lymphoid tissue. These cells most closely resembled effector memory T cells, usually associated with circulation through non-lymphoid tissue, but here, these cells were retained in the draining lymph node. These data indicate that lymphoid tissue resident memory CD4 + T-cell populations are generated in peripheral lymph nodes following immunisation. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

21 CFR 660.35 - Labeling.

Code of Federal Regulations, 2010 CFR

2010-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.35 Labeling. In... or end of the label, oustide of the main panel. (2) If washing the cells is required by the manufacturer, the container label shall include appropriate instructions; if the cells should not be washed...

In Vivo MR Imaging of Glioma Recruitment of Adoptive T-Cells Labeled with NaGdF4 -TAT Nanoprobes.

PubMed

Zhang, Hua; Wu, Yue; Wang, Jing; Tang, Zhongmin; Ren, Yan; Ni, Dalong; Gao, Hongbo; Song, Ruixue; Jin, Teng; Li, Qiao; Bu, Wenbo; Yao, Zhenwei

2018-01-01

Adoptive T lymphocyte immunotherapy is one of the most promising methods to treat residual lesions after glioma surgery. However, the fate of the adoptively transferred T-cells in vivo is unclear, hampering the understanding of this emerging therapy. Thus, it is highly desirable to develop noninvasive and quantitative in vivo tracking of these T-cells to glioma for better identification of the migratory fate and to provide objective evaluation of outcomes of adoptive T-cell immunotherapy targeting glioma. In this work, ultrasmall T 1 MR-based nanoprobes, NaGdF 4 -TAT, as molecular probes with high longitudinal relaxivity (8.93 mm -1 s -1 ) are designed. By means of HIV-1 transactivator (TAT) peptides, nearly 95% of the adoptive T-cells are labeled with the NaGdF 4 -TAT nanoprobes without any measurable side effects on the labeled T-cells, which is remarkably superior to that of the control fluorescein isothiocyanate-NaGdF 4 concerning labeling efficacy. Labeled adoptive T-cell clusters can be sensitively tracked in an orthotopic GL261-glioma model 24 h after intravenous infusion of 10 7 labeled T-cells by T 1 -weighted MR imaging. Both in vitro and in vivo experiments show that the NaGdF 4 -TAT nanoprobes labeling of T-cells may be a promising method to track adoptive T-cells to improve our understanding of the pathophysiology in adoptive immunotherapy for gliomas. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

NASA Astrophysics Data System (ADS)

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

2012-02-01

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

Cell-free measurements of brightness of fluorescently labeled antibodies

PubMed Central

Zhou, Haiying; Tourkakis, George; Shi, Dennis; Kim, David M.; Zhang, Hairong; Du, Tommy; Eades, William C.; Berezin, Mikhail Y.

2017-01-01

Validation of imaging contrast agents, such as fluorescently labeled imaging antibodies, has been recognized as a critical challenge in clinical and preclinical studies. As the number of applications for imaging antibodies grows, these materials are increasingly being subjected to careful scrutiny. Antibody fluorescent brightness is one of the key parameters that is of critical importance. Direct measurements of the brightness with common spectroscopy methods are challenging, because the fluorescent properties of the imaging antibodies are highly sensitive to the methods of conjugation, degree of labeling, and contamination with free dyes. Traditional methods rely on cell-based assays that lack reproducibility and accuracy. In this manuscript, we present a novel and general approach for measuring the brightness using antibody-avid polystyrene beads and flow cytometry. As compared to a cell-based method, the described technique is rapid, quantitative, and highly reproducible. The proposed method requires less than ten microgram of sample and is applicable for optimizing synthetic conjugation procedures, testing commercial imaging antibodies, and performing high-throughput validation of conjugation procedures. PMID:28150730

Chemical biology-based approaches on fluorescent labeling of proteins in live cells.

PubMed

Jung, Deokho; Min, Kyoungmi; Jung, Juyeon; Jang, Wonhee; Kwon, Youngeun

2013-05-01

Recently, significant advances have been made in live cell imaging owing to the rapid development of selective labeling of proteins in vivo. Green fluorescent protein (GFP) was the first example of fluorescent reporters genetically introduced to protein of interest (POI). While GFP and various types of engineered fluorescent proteins (FPs) have been actively used for live cell imaging for many years, the size and the limited windows of fluorescent spectra of GFP and its variants set limits on possible applications. In order to complement FP-based labeling methods, alternative approaches that allow incorporation of synthetic fluorescent probes to target POIs were developed. Synthetic fluorescent probes are smaller than fluorescent proteins, often have improved photochemical properties, and offer a larger variety of colors. These synthetic probes can be introduced to POIs selectively by numerous approaches that can be largely categorized into chemical recognition-based labeling, which utilizes metal-chelating peptide tags and fluorophore-carrying metal complexes, and biological recognition-based labeling, such as (1) specific non-covalent binding between an enzyme tag and its fluorophore-carrying substrate, (2) self-modification of protein tags using substrate variants conjugated to fluorophores, (3) enzymatic reaction to generate a covalent binding between a small molecule substrate and a peptide tag, and (4) split-intein-based C-terminal labeling of target proteins. The chemical recognition-based labeling reaction often suffers from compromised selectivity of metal-ligand interaction in the cytosolic environment, consequently producing high background signals. Use of protein-substrate interactions or enzyme-mediated reactions generally shows improved specificity but each method has its limitations. Some examples are the presence of large linker protein, restriction on the choice of introducible probes due to the substrate specificity of enzymes, and competitive

Confocal Fluorescence Imaging Enables Noninvasive Quantitative Assessment of Host Cell Populations In Vivo Following Photodynamic Therapy

PubMed Central

Mitra, Soumya; Mironov, Oleg; Foster, Thomas H.

2012-01-01

We report the use of optical imaging strategies to noninvasively examine photosensitizer distribution and physiological and host responses to 2-[1-hexyloxyethyl]-2 devinyl pyropheophorbide-a (HPPH)-mediated photodynamic therapy (PDT) of EMT6 tumors established in the ears of BALB/c mice. 24 h following intravenous (IV) administration of 1 μmol kg-1 HPPH, wide-field fluorescence imaging reveals tumor selectivity with an approximately 2-3-fold differential between tumor and adjacent normal tissue. Confocal microscopy demonstrates a relatively homogeneous intratumor HPPH distribution. Labeling of host cells using fluorophore-conjugated antibodies allowed the visualization of Gr1+/CD11b+ leukocytes and major histocompatibility complex class II (MHC-II)+ cells in vivo. Imaging of the treated site at different time-points following irradiation shows significant and rapid increases in Gr1+ cells in response to therapy. The maximum accumulation of Gr1+ cells is found at 24 h post-irradiation, followed by a decrease at the 48 h time-point. Using IV-injected FITC-conjugated dextran as a fluorescent perfusion marker, we imaged tissue perfusion at different times post-irradiation and found that the reduced Gr1+ cell density at 48 h correlated strongly with functional damage to the vasculature as reported via decreased perfusion status. Dual color confocal imaging experiments demonstrates that about 90% of the anti-Gr1 cell population co-localized with anti-CD11b labeling, thus indicating that majority of the Gr1-labeled cells were neutrophils. At 24 h post-PDT, an approximately 2-fold increase in MHC-II+ cells relative to untreated control is also observed. Co-localization analysis reveals an increase in the fraction of Gr1+ cells expressing MHC-II, suggesting that HPPH-PDT is stimulating neutrophils to express an antigen-presenting phenotype. PMID:23082097

Label free imaging of cell-substrate contacts by holographic total internal reflection microscopy.

PubMed

Mandracchia, Biagio; Gennari, Oriella; Marchesano, Valentina; Paturzo, Melania; Ferraro, Pietro

2017-09-01

The study of cell adhesion contacts is pivotal to understand cell mechanics and interaction at substrates or chemical and physical stimuli. We designed and built a HoloTIR microscope for label-free quantitative phase imaging of total internal reflection. Here we show for the first time that HoloTIR is a good choice for label-free study of focal contacts and of cell/substrate interaction as its sensitivity is enhanced in comparison with standard TIR microscopy. Finally, the simplicity of implementation and relative low cost, due to the requirement of less optical components, make HoloTIR a reasonable alternative, or even an addition, to TIRF microscopy for mapping cell/substratum topography. As a proof of concept, we studied the formation of focal contacts of fibroblasts on three substrates with different levels of affinity for cell adhesion. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

Actis, Paolo; Mak, Andy C; Pourmand, Nader

2010-08-01

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.

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

Size distribution of retrovirally marked lineages matches prediction from population measurements of cell cycle behavior

NASA Technical Reports Server (NTRS)

Cai, Li; Hayes, Nancy L.; Takahashi, Takao; Caviness, Verne S Jr; Nowakowski, Richard S.

2002-01-01

Mechanisms that regulate neuron production in the developing mouse neocortex were examined by using a retroviral lineage marking method to determine the sizes of the lineages remaining in the proliferating population of the ventricular zone during the period of neuron production. The distribution of clade sizes obtained experimentally in four different injection-survival paradigms (E11-E13, E11-E14, E11-E15, and E12-E15) from a total of over 500 labeled lineages was compared with that obtained from three models in which the average behavior of the proliferating population [i.e., the proportion of cells remaining in the proliferative population (P) vs. that exiting the proliferative population (Q)] was quantitatively related to lineage size distribution. In model 1, different proportions of asymmetric, symmetric terminal, and symmetric nonterminal cell divisions coexisted during the entire developmental period. In model 2, the developmental period was divided into two epochs: During the first, asymmetric and symmetric nonterminal cell divisions occurred, but, during the second, asymmetric and symmetric terminal cell divisions occurred. In model 3, the shifts in P and Q are accounted for by changes in the proportions of the two types of symmetric cell divisions without the inclusion of any asymmetric cell divisions. The results obtained from the retroviral experiments were well accounted for by model 1 but not by model 2 or 3. These findings demonstrate that: 1) asymmetric and both types of symmetric cell divisions coexist during the entire period of neurogenesis in the mouse, 2) neuron production is regulated in the proliferative population by the independent decisions of the two daughter cells to reenter S phase, and 3) neurons are produced by both asymmetric and symmetric terminal cell divisions. In addition, the findings mean that cell death and/or tangential movements of cells in the proliferative population occur at only a low rate and that there are no

Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope

PubMed Central

Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

2015-01-01

Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications. PMID:26525841

Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope.

PubMed

Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

2015-11-03

Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications.

Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope

NASA Astrophysics Data System (ADS)

Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

2015-11-01

Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications.

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

PubMed

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

2015-09-01

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

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

[131I]FIAU labeling of genetically transduced, tumor-reactive lymphocytes: cell-level dosimetry and dose-dependent toxicity.

PubMed

Zanzonico, Pat; Koehne, Guenther; Gallardo, Humilidad F; Doubrovin, Mikhail; Doubrovina, Ekaterina; Finn, Ronald; Blasberg, Ronald G; Riviere, Isabelle; O'Reilly, Richard J; Sadelain, Michel; Larson, Steven M

2006-09-01

Donor T cells have been shown to be reactive against and effective in adoptive immunotherapy of Epstein-Barr virus (EBV) lymphomas which develop in some leukemia patients post marrow transplantation. These T cells may be genetically modified by incorporation of a replication-incompetent viral vector (NIT) encoding both an inactive mutant nerve growth factor receptor (LNGFR), as an immunoselectable surface marker, and a herpes simplex virus thymidine kinase (HSV-TK), rendering the cells sensitive to ganciclovir. The current studies are based on the selective HSV-TK-catalyzed trapping (phosphorylation) of the thymidine analog [(131)I]-2'-fluoro-2'-deoxy-1-beta-D-arabinofuransyl-5-iodo-uracil (FIAU) as a means of stably labeling such T cells for in vivo trafficking (including tumor targeting) studies. Because of the radiosensitivity of lymphocytes and the potentially high absorbed dose to the nucleus from intracellular (131)I (even at tracer levels), the nucleus absorbed dose (D ( n )) and dose-dependent immune functionality were evaluated for NIT(+) T cells labeled ex vivo in [(131)I]FIAU-containing medium. Based on in vitro kinetic studies of [(131)I]FIAU uptake by NIT(+) T cells, D ( n ) was calculated using an adaptation of the MIRD formalism and the recently published MIRD cellular S factors. Immune cytotoxicity of [(131)I]FIAU-labeled cells was assayed against (51)Cr-labeled target cells [B-lymphoblastoid cells (BLCLs)] in a standard 4-h release assay. At median nuclear absorbed doses up to 830 cGy, a (51)Cr-release assay against BLCLs showed no loss of immune cytotoxicity, thus demonstrating the functional integrity of genetically transduced, tumor-reactive T cells labeled at this dose level for in vivo cell trafficking and tumor targeting studies.

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

PubMed Central

Provost, Christopher R.; Sun, Luo

2010-01-01

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells. PMID:20485262

Bone marrow cells stained by azide-conjugated Alexa fluors in the absence of an alkyne label.

PubMed

Lin, Guiting; Ning, Hongxiu; Banie, Lia; Qiu, Xuefeng; Zhang, Haiyang; Lue, Tom F; Lin, Ching-Shwun

2012-09-01

Thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) has recently been introduced as an alternative to 5-bromo-2-deoxyuridine (BrdU) for cell labeling and tracking. Incorporation of EdU into replicating DNA can be detected by azide-conjugated fluors (eg, Alexa-azide) through a Cu(i)-catalyzed click reaction between EdU's alkyne moiety and azide. While this cell labeling method has proven to be valuable for tracking transplanted stem cells in various tissues, we have found that some bone marrow cells could be stained by Alexa-azide in the absence of EdU label. In intact rat femoral bone marrow, ~3% of nucleated cells were false-positively stained, and in isolated bone marrow cells, ~13%. In contrast to true-positive stains, which localize in the nucleus, the false-positive stains were cytoplasmic. Furthermore, while true-positive staining requires Cu(i), false-positive staining does not. Reducing the click reaction time or reducing the Alexa-azide concentration failed to improve the distinction between true- and false-positive staining. Hematopoietic and mesenchymal stem cell markers CD34 and Stro-1 did not co-localize with the false-positively stained cells, and these cells' identity remains unknown.

Regional differences in endothelial cell cytoskeleton, junctional proteins and phosphorylated tyrosine labeling in the porcine vortex vein system.

PubMed

Tan, Priscilla Ern Zhi; Yu, Paula K; Yang, Hongfang; Cringle, Stephen J; Yu, Dao-Yi

2018-07-01

We previously demonstrated endothelial phenotype heterogeneity in the vortex vein system. This study is to further determine whether regional differences are present in the cytoskeleton, junctional proteins and phosphorylated tyrosine labeling within the system. The vortex vein system of twenty porcine eyes was perfused with labels for f-actin, claudin-5, VE-Cadherin, phosphorylated tyrosine and nucleic acid. The endothelial cells of eight different regions (choroidal veins, pre-ampulla, anterior ampulla, mid-ampulla, posterior ampulla, post-ampulla, intra-scleral canal and the extra-ocular vortex vein) were studied using confocal microscopy. There were regional differences in the endothelial cell structures. Cytoskeleton labeling was relatively even in intensity throughout Regions 1 to 6. Overall VE-Cadherin had a non-uniform distribution and thicker width endothelial cell border staining than claudin-5. Progressing downstream there was an increased variation in thickness of VE-cadherin labeling. There was an overlap in phosphorylated tyrosine and VE-Cadherin labeling in the post-ampulla, intra-scleral canal and extra-ocular vortex vein. Intramural cells were observed that were immune-positive for VE-Cadherin and phosphorylated tyrosine. There were significant differences in the number of intramural cells in different regions. Significant regional differences with endothelial cell labeling of cytoskeleton, junction proteins, and phosphorylated tyrosine were found within the vortex vein system. These findings support existing data on endothelial cell phenotype heterogeneity, and may aid in the knowledge of venous pathologies by understanding regions of vulnerability to endothelial damage within the vortex vein system. It could be valuable to further investigate and characterize the VE-cadherin and phosphotyrosine immune-positive intramural cells. Copyright © 2018. Published by Elsevier Ltd.

Behaviour of adipose-derived canine mesenchymal stem cells after superparamagnetic iron oxide nanoparticles labelling for magnetic resonance imaging.

PubMed

Kolecka, Malgorzata Anna; Arnhold, Stefan; Schmidt, Martin; Reich, Christine; Kramer, Martin; Failing, Klaus; von Pückler, Kerstin

2017-02-24

Therapy with mesenchymal stem cells (MSCs) has been reported to provide beneficial effects in the treatment of neurological and orthopaedic disorders in dogs. The exact mechanism of action is poorly understood. Magnetic resonance imaging (MRI) gives the opportunity to observe MSCs after clinical administration. To visualise MSCs with the help of MRI, labelling with an MRI contrast agent is necessary. However, it must be clarified whether there is any negative influence on cell function and viability after labelling prior to clinical administration. For the purpose of the study, seven samples with canine adipose-derived stem cells were incubated with superparamagnetic iron oxide nanoparticles (SPIO: 319.2 μg/mL Fe) for 24 h. The internalisation of the iron particles occurred via endocytosis. SPIO particles were localized as free clusters in the cytoplasm or within lysosomes depending on the time of investigation. The efficiency of the labelling was investigated using Prussian blue staining and MACS assay. After 3 weeks the percentage of SPIO labelled canine stem cells decreased. Phalloidin staining showed no negative effect on the cytoskeleton. Labelled cells underwent osteogenic and adipogenic differentiation. Chondrogenic differentiation occurred to a lesser extent compared with a control sample. MTT-Test and wound healing assay showed no influence of labelling on the proliferation. The duration of SPIO labelling was assessed using a 1 Tesla clinical MRI scanner and T2 weighted turbo spin echo and T2 weighted gradient echo MRI sequences 1, 2 and 3 weeks after labelling. The hypointensity caused by SPIO lasted for 3 weeks in both sequences. An Endorem labelling concentration of 319.2 μg/mL Fe (448 μg/mL SPIO) had no adverse effects on the viability of canine ASCs. Therefore, this contrast agent could be used as a model for iron oxide labelling agents. However, the tracking ability in vivo has to be evaluated in further studies.

Poly (dopamine) coated superparamagnetic iron oxide nanocluster for noninvasive labeling, tracking, and targeted delivery of adipose tissue-derived stem cells.

PubMed

Liao, Naishun; Wu, Ming; Pan, Fan; Lin, Jiumao; Li, Zuanfang; Zhang, Da; Wang, Yingchao; Zheng, Youshi; Peng, Jun; Liu, Xiaolong; Liu, Jingfeng

2016-01-05

Tracking and monitoring of cells in vivo after transplantation can provide crucial information for stem cell therapy. Magnetic resonance imaging (MRI) combined with contrast agents is believed to be an effective and non-invasive technique for cell tracking in living bodies. However, commercial superparamagnetic iron oxide nanoparticles (SPIONs) applied to label cells suffer from shortages such as potential toxicity, low labeling efficiency, and low contrast enhancing. Herein, the adipose tissue-derived stem cells (ADSCs) were efficiently labeled with SPIONs coated with poly (dopamine) (SPIONs cluster@PDA), without affecting their viability, proliferation, apoptosis, surface marker expression, as well as their self-renew ability and multi-differentiation potential. The labeled cells transplanted into the mice through tail intravenous injection exhibited a negative enhancement of the MRI signal in the damaged liver-induced by carbon tetrachloride, and subsequently these homed ADSCs with SPIONs cluster@PDA labeling exhibited excellent repair effects to the damaged liver. Moreover, the enhanced target-homing to tissue of interest and repair effects of SPIONs cluster@PDA-labeled ADSCs could be achieved by use of external magnetic field in the excisional skin wound mice model. Therefore, we provide a facile, safe, noninvasive and sensitive method for external magnetic field targeted delivery and MRI based tracking of transplanted cells in vivo.

Poly (dopamine) coated superparamagnetic iron oxide nanocluster for noninvasive labeling, tracking, and targeted delivery of adipose tissue-derived stem cells

PubMed Central

Liao, Naishun; Wu, Ming; Pan, Fan; Lin, Jiumao; Li, Zuanfang; Zhang, Da; Wang, Yingchao; Zheng, Youshi; Peng, Jun; Liu, Xiaolong; Liu, Jingfeng

2016-01-01

Tracking and monitoring of cells in vivo after transplantation can provide crucial information for stem cell therapy. Magnetic resonance imaging (MRI) combined with contrast agents is believed to be an effective and non-invasive technique for cell tracking in living bodies. However, commercial superparamagnetic iron oxide nanoparticles (SPIONs) applied to label cells suffer from shortages such as potential toxicity, low labeling efficiency, and low contrast enhancing. Herein, the adipose tissue-derived stem cells (ADSCs) were efficiently labeled with SPIONs coated with poly (dopamine) (SPIONs cluster@PDA), without affecting their viability, proliferation, apoptosis, surface marker expression, as well as their self-renew ability and multi-differentiation potential. The labeled cells transplanted into the mice through tail intravenous injection exhibited a negative enhancement of the MRI signal in the damaged liver-induced by carbon tetrachloride, and subsequently these homed ADSCs with SPIONs cluster@PDA labeling exhibited excellent repair effects to the damaged liver. Moreover, the enhanced target-homing to tissue of interest and repair effects of SPIONs cluster@PDA-labeled ADSCs could be achieved by use of external magnetic field in the excisional skin wound mice model. Therefore, we provide a facile, safe, noninvasive and sensitive method for external magnetic field targeted delivery and MRI based tracking of transplanted cells in vivo. PMID:26728448

Poly (dopamine) coated superparamagnetic iron oxide nanocluster for noninvasive labeling, tracking, and targeted delivery of adipose tissue-derived stem cells

NASA Astrophysics Data System (ADS)

Liao, Naishun; Wu, Ming; Pan, Fan; Lin, Jiumao; Li, Zuanfang; Zhang, Da; Wang, Yingchao; Zheng, Youshi; Peng, Jun; Liu, Xiaolong; Liu, Jingfeng

2016-01-01

Tracking and monitoring of cells in vivo after transplantation can provide crucial information for stem cell therapy. Magnetic resonance imaging (MRI) combined with contrast agents is believed to be an effective and non-invasive technique for cell tracking in living bodies. However, commercial superparamagnetic iron oxide nanoparticles (SPIONs) applied to label cells suffer from shortages such as potential toxicity, low labeling efficiency, and low contrast enhancing. Herein, the adipose tissue-derived stem cells (ADSCs) were efficiently labeled with SPIONs coated with poly (dopamine) (SPIONs cluster@PDA), without affecting their viability, proliferation, apoptosis, surface marker expression, as well as their self-renew ability and multi-differentiation potential. The labeled cells transplanted into the mice through tail intravenous injection exhibited a negative enhancement of the MRI signal in the damaged liver-induced by carbon tetrachloride, and subsequently these homed ADSCs with SPIONs cluster@PDA labeling exhibited excellent repair effects to the damaged liver. Moreover, the enhanced target-homing to tissue of interest and repair effects of SPIONs cluster@PDA-labeled ADSCs could be achieved by use of external magnetic field in the excisional skin wound mice model. Therefore, we provide a facile, safe, noninvasive and sensitive method for external magnetic field targeted delivery and MRI based tracking of transplanted cells in vivo.

Flow-Mediated Stem Cell Labeling with Superparamagnetic Iron Oxide Nanoparticle Clusters

PubMed Central

Shkumatov, Artem; Lai, Mei-Hsiu; Smith, Cartney E.; Rich, Max; Kong, Hyunjoon

2013-01-01

This study presents a strategy to enhance the uptake of superparamagnetic iron oxide nanoparticle (SPIO) clusters by manipulating the cellular mechanical environment. Specifically, stem cells exposed to an orbital flow ingested almost two-fold greater amount of SPIO clusters than those cultured statically. Improvements in MR contrast were subsequently achieved for labeled cells in collagen gels and a mouse model. Overall, this strategy will serve to improve the efficiency of cell tracking and therapies. PMID:24033276

Biocompatible and label-free separation of cancer cells from cell culture lines from white blood cells in ferrofluids.

PubMed

Zhao, Wujun; Cheng, Rui; Lim, So Hyun; Miller, Joshua R; Zhang, Weizhong; Tang, Wei; Xie, Jin; Mao, Leidong

2017-06-27

This paper reports a biocompatible and label-free cell separation method using ferrofluids that can separate a variety of low-concentration cancer cells from cell culture lines (∼100 cancer cells per mL) from undiluted white blood cells, with a throughput of 1.2 mL h -1 and an average separation efficiency of 82.2%. The separation is based on the size difference of the cancer cells and white blood cells, and is conducted in a custom-made biocompatible ferrofluid that retains not only excellent short-term viabilities but also normal proliferations of 7 commonly used cancer cell lines. A microfluidic device is designed and optimized specifically to shorten the time of live cells' exposure to ferrofluids from hours to seconds, by eliminating time-consuming off-chip sample preparation and extraction steps and integrating them on-chip to achieve a one-step process. As a proof-of-concept demonstration, a ferrofluid with 0.26% volume fraction was used in this microfluidic device to separate spiked cancer cells from cell lines at a concentration of ∼100 cells per mL from white blood cells with a throughput of 1.2 mL h -1 . The separation efficiencies were 80 ± 3%, 81 ± 5%, 82 ± 5%, 82 ± 4%, and 86 ± 6% for A549 lung cancer, H1299 lung cancer, MCF-7 breast cancer, MDA-MB-231 breast cancer, and PC-3 prostate cancer cell lines, respectively. The separated cancer cells' purity was between 25.3% and 28.8%. In addition, the separated cancer cells from this strategy showed an average short-term viability of 94.4 ± 1.3%, and these separated cells were cultured and demonstrated normal proliferation to confluence even after the separation process. Owing to its excellent biocompatibility and label-free operation and its ability to recover low concentrations of cancer cells from white blood cells, this method could lead to a promising tool for rare cell separation.

Simple SPION Incubation as an Efficient Intracellular Labeling Method for Tracking Neural Progenitor Cells Using MRI

PubMed Central

D. M., Jayaseema; Lai, Jiann-Shiun; Hueng, Dueng-Yuan; Chang, Chen

2013-01-01

Cellular magnetic resonance imaging (MRI) has been well-established for tracking neural progenitor cells (NPC). Superparamagnetic iron oxide nanoparticles (SPIONs) approved for clinical application are the most common agents used for labeling. Conventionally, transfection agents (TAs) were added with SPIONs to facilitate cell labeling because SPIONs in the native unmodified form were deemed inefficient for intracellular labeling. However, compelling evidence also shows that simple SPION incubation is not invariably ineffective. The labeling efficiency can be improved by prolonged incubation and elevated iron doses. The goal of the present study was to establish simple SPION incubation as an efficient intracellular labeling method. To this end, NPCs derived from the neonatal subventricular zone were incubated with SPIONs (Feridex®) and then evaluated in vitro with regard to the labeling efficiency and biological functions. The results showed that, following 48 hours of incubation at 75 µg/ml, nearly all NPCs exhibited visible SPION intake. Evidence from light microscopy, electron microscopy, chemical analysis, and magnetic resonance imaging confirmed the effectiveness of the labeling. Additionally, biological assays showed that the labeled NPCs exhibited unaffected viability, oxidative stress, apoptosis and differentiation. In the demonstrated in vivo cellular MRI experiment, the hypointensities representing the SPION labeled NPCs remained observable throughout the entire tracking period. The findings indicate that simple SPION incubation without the addition of TAs is an efficient intracellular magnetic labeling method. This simple approach may be considered as an alternative approach to the mainstream labeling method that involves the use of TAs. PMID:23468856

High-throughput, label-free, single-cell, microalgal lipid screening by machine-learning-equipped optofluidic time-stretch quantitative phase microscopy.

PubMed

Guo, Baoshan; Lei, Cheng; Kobayashi, Hirofumi; Ito, Takuro; Yalikun, Yaxiaer; Jiang, Yiyue; Tanaka, Yo; Ozeki, Yasuyuki; Goda, Keisuke

2017-05-01

The development of reliable, sustainable, and economical sources of alternative fuels to petroleum is required to tackle the global energy crisis. One such alternative is microalgal biofuel, which is expected to play a key role in reducing the detrimental effects of global warming as microalgae absorb atmospheric CO 2 via photosynthesis. Unfortunately, conventional analytical methods only provide population-averaged lipid amounts and fail to characterize a diverse population of microalgal cells with single-cell resolution in a non-invasive and interference-free manner. Here high-throughput label-free single-cell screening of lipid-producing microalgal cells with optofluidic time-stretch quantitative phase microscopy was demonstrated. In particular, Euglena gracilis, an attractive microalgal species that produces wax esters (suitable for biodiesel and aviation fuel after refinement), within lipid droplets was investigated. The optofluidic time-stretch quantitative phase microscope is based on an integration of a hydrodynamic-focusing microfluidic chip, an optical time-stretch quantitative phase microscope, and a digital image processor equipped with machine learning. As a result, it provides both the opacity and phase maps of every single cell at a high throughput of 10,000 cells/s, enabling accurate cell classification without the need for fluorescent staining. Specifically, the dataset was used to characterize heterogeneous populations of E. gracilis cells under two different culture conditions (nitrogen-sufficient and nitrogen-deficient) and achieve the cell classification with an error rate of only 2.15%. The method holds promise as an effective analytical tool for microalgae-based biofuel production. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Binding effect of fluorescence labeled glycyrrhetinic acid with GA receptors in hepatocellular carcinoma cells.

PubMed

Sun, Yu-Qi; Dai, Chun-Mei; Zheng, Yan; Shi, Shu-Dan; Hu, Hai-Yang; Chen, Da-Wei

2017-11-01

Glycyrrhetinic acid (GA) is a natural active component from licorice, which is broadly used in traditional Chinese medicine. Lots of glycyrrhetinic acid receptors (GA-R) are proved to locate on the surface of liver cells. Many reports about the hepatocellular carcinoma (HCC) treatment were dependent on GA modified carriers. However, the reality of GA-R in HCC cells was not clear. In this paper, 18β-glycyrrhetinic acid (18β-GA) was labeled with fluorescence (FITC) by chemical synthesis. Together with the binding effect of fluorescence labeled glycyrrhetinic acid (FITC-GA), the competitive action of 18β-GA with GA-R was investigated in HCC cells. The results showed that in HepG2 cells, 18β-GA and FITC-GA presented similar cytotoxicity. The specific binding saturation of GA showed the dissociation constant (K d ) was 7.457±2.122pmol/L and the maximum binding counts (B max ) was 2.385±0.175pmol/2.5×10 6 cells, respectively. FITC-GA bound to cytomembrane specifically and 18β-GA competed to bind the sites significantly in HepG2 cells. Therefore, there is binding effect between fluorescence labeled GA and GA-R. The GA-R on HCC cells is confirmed as expected, which provides a useful reference of active target modified by GA and a novel approach for receptors and ligands study. Copyright © 2017 Elsevier Inc. All rights reserved.

Detection of intramyocardially injected DiR-labeled mesenchymal stem cells by optical and optoacoustic tomography.

PubMed

Berninger, Markus T; Mohajerani, Pouyan; Wildgruber, Moritz; Beziere, Nicolas; Kimm, Melanie A; Ma, Xiaopeng; Haller, Bernhard; Fleming, Megan J; Vogt, Stephan; Anton, Martina; Imhoff, Andreas B; Ntziachristos, Vasilis; Meier, Reinhard; Henning, Tobias D

2017-06-01

The distribution of intramyocardially injected rabbit MSCs, labeled with the near-infrared dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbo-cyanine-iodide (DiR) using hybrid Fluorescence Molecular Tomography-X-ray Computed Tomography (FMT-XCT) and Multispectral Optoacoustic Tomography (MSOT) imaging technologies, was investigated. Viability and induction of apoptosis of DiR labeled MSCs were assessed by XTT- and Caspase-3/-7-testing in vitro . 2 × 10 6 , 2 × 10 5 and 2 × 10 4 MSCs labeled with 5 and 10 μg DiR/ml were injected into fresh frozen rabbit hearts. FMT-XCT, MSOT and fluorescence cryosection imaging were performed. Concentrations up to 10 μg DiR/ml did not cause apoptosis in vitro (p > 0.05). FMT and MSOT imaging of labeled MSCs led to a strong signal. The imaging modalities highlighted a difference in cell distribution and concentration correlated to the number of injected cells. Ex-vivo cryosectioning confirmed the molecular fluorescence signal. FMT and MSOT are sensitive imaging techniques offering high-anatomic resolution in terms of detection and distribution of intramyocardially injected stem cells in a rabbit model.

Recyclable Cu(i)/melanin dots for cycloaddition, bioconjugation and cell labelling

DOE PAGES

Sun, Yao; Hong, Suhyun; Ma, Xiaowei; ...

2016-05-20

We successfully transferred melanin into a novel catalytic platform. Ligand-free, water-soluble, recyclable and biocompatible Cu(i)-loaded melanin dots [Cu(i)/M-dots] was easily prepared and demonstrate excellent properties for classic CuAAC, bioconjugation and cell labelling.

Label-free cell-cycle analysis by high-throughput quantitative phase time-stretch imaging flow cytometry

NASA Astrophysics Data System (ADS)

Mok, Aaron T. Y.; Lee, Kelvin C. M.; Wong, Kenneth K. Y.; Tsia, Kevin K.

2018-02-01

Biophysical properties of cells could complement and correlate biochemical markers to characterize a multitude of cellular states. Changes in cell size, dry mass and subcellular morphology, for instance, are relevant to cell-cycle progression which is prevalently evaluated by DNA-targeted fluorescence measurements. Quantitative-phase microscopy (QPM) is among the effective biophysical phenotyping tools that can quantify cell sizes and sub-cellular dry mass density distribution of single cells at high spatial resolution. However, limited camera frame rate and thus imaging throughput makes QPM incompatible with high-throughput flow cytometry - a gold standard in multiparametric cell-based assay. Here we present a high-throughput approach for label-free analysis of cell cycle based on quantitative-phase time-stretch imaging flow cytometry at a throughput of > 10,000 cells/s. Our time-stretch QPM system enables sub-cellular resolution even at high speed, allowing us to extract a multitude (at least 24) of single-cell biophysical phenotypes (from both amplitude and phase images). Those phenotypes can be combined to track cell-cycle progression based on a t-distributed stochastic neighbor embedding (t-SNE) algorithm. Using multivariate analysis of variance (MANOVA) discriminant analysis, cell-cycle phases can also be predicted label-free with high accuracy at >90% in G1 and G2 phase, and >80% in S phase. We anticipate that high throughput label-free cell cycle characterization could open new approaches for large-scale single-cell analysis, bringing new mechanistic insights into complex biological processes including diseases pathogenesis.

Hopx expression defines a subset of multipotent hair follicle stem cells and a progenitor population primed to give rise to K6+ niche cells

PubMed Central

Takeda, Norifumi; Jain, Rajan; LeBoeuf, Matthew R.; Padmanabhan, Arun; Wang, Qiaohong; Li, Li; Lu, Min Min; Millar, Sarah E.; Epstein, Jonathan A.

2013-01-01

The mammalian hair follicle relies on adult resident stem cells and their progeny to fuel and maintain hair growth throughout the life of an organism. The cyclical and initially synchronous nature of hair growth makes the hair follicle an ideal system with which to define homeostatic mechanisms of an adult stem cell population. Recently, we demonstrated that Hopx is a specific marker of intestinal stem cells. Here, we show that Hopx specifically labels long-lived hair follicle stem cells residing in the telogen basal bulge. Hopx+ cells contribute to all lineages of the mature hair follicle and to the interfollicular epidermis upon epidermal wounding. Unexpectedly, our analysis identifies a previously unappreciated progenitor population that resides in the lower hair bulb of anagen-phase follicles and expresses Hopx. These cells co-express Lgr5, do not express Shh and escape catagen-induced apoptosis. They ultimately differentiate into the cytokeratin 6-positive (K6) inner bulge cells in telogen, which regulate the quiescence of adjacent hair follicle stem cells. Although previous studies have suggested that K6+ cells arise from Lgr5-expressing lower outer root sheath cells in anagen, our studies indicate an alternative origin, and a novel role for Hopx-expressing lower hair bulb progenitor cells in contributing to stem cell homeostasis. PMID:23487314

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

PubMed

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

2016-05-18

Mesenchymal stem cells (MSCs) are among the major stem cells used for cell therapy and regenerative medicine. In-vivo cell-tracking by magnetic resonance imaging (MRI) is crucial for regenerative medicine, allowing verification that the transplanted cells reach the targeted sites. Cellular MRI combined with superparamagnetic iron-oxide (SPIO) contrast agents is an effective cell-tracking method. Here, we are reporting a new "bio-mimicry" method by making use of the "in-vivo environment" of MSCs to prepare native MSCs, so that (i) the phagocytic activity of cultured MSCs can be recovered and expanded MSCs can be ex-vivo labeled with Ferumoxytol, which is currently the only FDA approved SPIO nanoparticles for human use. Using our new method, 7-day cultured MSCs regain the capability to take up Ferumoxytol and exhibit an intracellular iron concentration of 2.50 ± 0.50 pg/MSC, comparable to that obtained by using Ferumoxytol-heparin-protamine nanocomplex; and (ii) cells can be re-sized to more native size, reducing from 32.0 ± 7.2 μm to 19.5 ± 5.2 μm. Our method can be very useful for expanding MSCs and labeling with Ferumoxytol, without the need for transfection agents and/or electroporation, allowing cell-tracking by MRI in both pre-clinical and clinical studies.

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

PubMed Central

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

2016-01-01

Mesenchymal stem cells (MSCs) are among the major stem cells used for cell therapy and regenerative medicine. In-vivo cell-tracking by magnetic resonance imaging (MRI) is crucial for regenerative medicine, allowing verification that the transplanted cells reach the targeted sites. Cellular MRI combined with superparamagnetic iron-oxide (SPIO) contrast agents is an effective cell-tracking method. Here, we are reporting a new “bio-mimicry” method by making use of the “in-vivo environment” of MSCs to prepare native MSCs, so that (i) the phagocytic activity of cultured MSCs can be recovered and expanded MSCs can be ex-vivo labeled with Ferumoxytol, which is currently the only FDA approved SPIO nanoparticles for human use. Using our new method, 7-day cultured MSCs regain the capability to take up Ferumoxytol and exhibit an intracellular iron concentration of 2.50 ± 0.50 pg/MSC, comparable to that obtained by using Ferumoxytol-heparin-protamine nanocomplex; and (ii) cells can be re-sized to more native size, reducing from 32.0 ± 7.2 μm to 19.5 ± 5.2 μm. Our method can be very useful for expanding MSCs and labeling with Ferumoxytol, without the need for transfection agents and/or electroporation, allowing cell-tracking by MRI in both pre-clinical and clinical studies. PMID:27188664

Cellulose and pectin localization in roots of mycorrhizalAllium porrum: labelling continuity between host cell wall and interfacial material.

PubMed

Bonfante-Fasolo, P; Vian, B; Perotto, S; Faccio, A; Knox, J P

1990-03-01

Two different types of contacts (or interfaces) exist between the plant host and the fungus during the vesicular-arbuscular mycorrhizal symbiosis, depending on whether the fungus is intercellular or intracellular. In the first case, the walls of the partners are in contact, while in the second case the fungal wall is separated from the host cytoplasm by the invaginated host plasmamembrane and by an interfacial material. In order to verify the origin of the interfacial material, affinity techniques which allow identification in situ of cell-wall components, were used. Cellobiohydrolase (CBH I) that binds to cellulose and a monoclonal antibody (JIM 5) that reacts with pectic components were tested on roots ofAllium porrum L. (leek) colonized byGlomus versiforme (Karst.) Berch. Both probes gave a labelling specific for the host cell wall, but each probe labelled over specific and distinct areas. The CBH I-colloidal gold complex heavily labelled the thick epidermal cell walls, whereas JIM 5 only labelled this area weakly. Labelling of the hypodermis was mostly on intercellular material after treatment with JIM 5 and only on the wall when CBH I was used. Suberin bands found on the radial walls were never labelled. Cortical cells were mostly labelled on the middle lamella with JIM 5 and on the wall with CBH I. Gold granules from the two probes were found in interfacial material both near the point where the fungus enters the cell and around the thin hyphae penetrating deep into the cell. The ultrastructural observations demonstrate that cellulose and pectic components have different but complementary distributions in the walls of root cells involved in the mycorrhizal symbiosis. These components show a similar distribution in the interfacial material laid down around the vesicular-arbuscular mycorrhizal fungus indicating that the interfacial material is of host origin.

Label-free in situ Imaging of Lignification in Plant Cell Walls

PubMed Central

Schmidt, Martin; Perera, Pradeep; Schwartzberg, Adam M.; Adams, Paul D.; Schuck, P. James

2010-01-01

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find cost-effective and sustainable solutions has become a topical and critical task. Lignocellulosic biomass is poised to become the primary source of biomass for the conversion to liquid biofuels1-6. However, the recalcitrance of these plant cell wall materials to cost-effective and efficient degradation presents a major impediment for their use in the production of biofuels and chemicals4. In particular, lignin, a complex and irregular poly-phenylpropanoid heteropolymer, becomes problematic to the postharvest deconstruction of lignocellulosic biomass. For example in biomass conversion for biofuels, it inhibits saccharification in processes aimed at producing simple sugars for fermentation7. The effective use of plant biomass for industrial purposes is in fact largely dependent on the extent to which the plant cell wall is lignified. The removal of lignin is a costly and limiting factor8 and lignin has therefore become a key plant breeding and genetic engineering target in order to improve cell wall conversion. Analytical tools that permit the accurate rapid characterization of lignification of plant cell walls become increasingly important for evaluating a large number of breeding populations. Extractive procedures for the isolation of native components such as lignin are inevitably destructive, bringing about significant chemical and structural modifications9-11. Analytical chemical in situ methods are thus invaluable tools for the compositional and structural characterization of lignocellulosic materials. Raman microscopy is a technique that relies on inelastic or Raman scattering of monochromatic light, like that from a laser, where the shift in energy of the laser photons is related to molecular vibrations and presents an intrinsic label-free molecular "fingerprint" of the sample. Raman microscopy can afford non

Physical labeling of papillomavirus-infected, immortal, and cancerous cervical epithelial cells reveal surface changes at immortal stage.

PubMed

Swaminathan Iyer, K; Gaikwad, R M; Woodworth, C D; Volkov, D O; Sokolov, Igor

2012-06-01

A significant change of surface features of malignant cervical epithelial cells compared to normal cells has been previously reported. Here, we are studying the question at which progressive stage leading to cervical cancer the surface alteration happens. A non-traditional method to identify malignant cervical epithelial cells in vitro, which is based on physical (in contrast to specific biochemical) labelling of cells with fluorescent silica micron-size beads, is used here to examine cells at progressive stages leading to cervical cancer which include normal epithelial cells, cells infected with human papillomavirus type-16 (HPV-16), cells immortalized by HPV-16, and carcinoma cells. The study shows a statistically significant (at p < 0.01) difference between both immortal and cancer cells and a group consisting of normal and infected. There is no significant difference between normal and infected cells. Immortal cells demonstrate the signal which is closer to cancer cells than to either normal or infected cells. This implies that the cell surface, surface cellular brush changes substantially when cells become immortal. Physical labeling of the cell surface represents a substantial departure from the traditional biochemical labeling methods. The results presented show the potential significance of physical properties of the cell surface for development of clinical methods for early detection of cervical cancer, even at the stage of immortalized, premalignant cells.

Physical Labeling of Papillomavirus-Infected, Immortal, and Cancerous Cervical Epithelial Cells Reveal Surface Changes at Immortal Stage

PubMed Central

Iyer, K. Swaminathan; Gaikwad, R. M.; Woodworth, C. D.; Volkov, D. O.

2013-01-01

A significant change of surface features of malignant cervical epithelial cells compared to normal cells has been previously reported. Here, we are studying the question at which progressive stage leading to cervical cancer the surface alteration happens. A non-traditional method to identify malignant cervical epithelial cells in vitro, which is based on physical (in contrast to specific biochemical) labelling of cells with fluorescent silica micron-size beads, is used here to examine cells at progressive stages leading to cervical cancer which include normal epithelial cells, cells infected with human papillomavirus type-16 (HPV-16), cells immortalized by HPV-16, and carcinoma cells. The study shows a statistically significant (at p <0.01) difference between both immortal and cancer cells and a group consisting of normal and infected. There is no significant difference between normal and infected cells. Immortal cells demonstrate the signal which is closer to cancer cells than to either normal or infected cells. This implies that the cell surface, surface cellular brush changes substantially when cells become immortal. Physical labeling of the cell surface represents a substantial departure from the traditional biochemical labeling methods. The results presented show the potential significance of physical properties of the cell surface for development of clinical methods for early detection of cervical cancer, even at the stage of immortalized, pre-malignant cells. PMID:22351422

Label-free in vitro prostate cancer cell detection via photonic-crystal biosensor

NASA Astrophysics Data System (ADS)

DeLuna, Frank; Ding, XiaoFei; Sagredo, Ismael; Bustamante, Gilbert; Sun, Lu-Zhe; Ye, Jing Yong

2018-02-01

Prostate-specific antigen (PSA) biomarker assays are the current clinical method for mass screening of prostate cancer. However, high false-positive rates are often reported due to PSA's low specificity, leading to an urgent need for the development of a more specific detection system independent of PSA levels. In our previous research, we demonstrated the feasibility of using cellular refractive indices (RI) as a unique contrast parameter to accomplish label-free detection of prostate cancer cells via variance testing, but were unable to determine if a specific cell was cancerous or noncancerous. In this paper, we report the use of our Photonic-Crystal biosensor in a Total-Internal-Reflection (PC-TIR) configuration to construct a label-free imaging system, which allows for the detection of individual prostate cancer cells utilizing cellular RI as the only contrast parameter. Noncancerous prostate (BPH-1) cells and prostate cancer (PC-3) cells were mixed at varied ratios and measured concurrently. Additionally, we isolated and induced PC-3 cells to undergo epithelial-mesenchymal transition (EMT) by exposing these cells to soluble factors such as TGF-β1. The biophysical characteristics of the cellular RI were quantified extensively in comparison to non-induced PC-3 cells as well as BPH-1 cells. EMT is a crucial mechanism for the invasion and metastasis of epithelial tumors characterized by the loss of cell-cell adhesion and increased cell mobility. Our study shows promising clinical potential in utilizing the PC-TIR biosensor imaging system to not only detect prostate cancer cells, but also evaluate prostate cancer progression.

Wide-field imaging and flow cytometric analysis of cancer cells in blood by fluorescent nanodiamond labeling and time gating

NASA Astrophysics Data System (ADS)

Hui, Yuen Yung; Su, Long-Jyun; Chen, Oliver Yenjyh; Chen, Yit-Tsong; Liu, Tzu-Ming; Chang, Huan-Cheng

2014-07-01

Nanodiamonds containing high density ensembles of negatively charged nitrogen-vacancy (NV-) centers are promising fluorescent biomarkers due to their excellent photostability and biocompatibility. The NV- centers in the particles have a fluorescence lifetime of up to 20 ns, which distinctly differs from those (<10 ns) of cell and tissue autofluorescence, making it possible to achieve background-free detection in vivo by time gating. Here, we demonstrate the feasibility of using fluorescent nanodiamonds (FNDs) as optical labels for wide-field time-gated fluorescence imaging and flow cytometric analysis of cancer cells with a nanosecond intensified charge-coupled device (ICCD) as the detector. The combined technique has allowed us to acquire fluorescence images of FND-labeled HeLa cells in whole blood covered with a chicken breast of ~0.1-mm thickness at the single cell level, and to detect individual FND-labeled HeLa cells in blood flowing through a microfluidic device at a frame rate of 23 Hz, as well as to locate and trace FND-labeled lung cancer cells in the blood vessels of a mouse ear. It opens a new window for real-time imaging and tracking of transplanted cells (such as stem cells) in vivo.

Double labeling of human leukemic cells using /sup 3/H-cytarabine and monoclonal antibody against bromodeoxyuridine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Raza, A.; Preisler, H.D.

A new technique using immunofluorescence and autoradiography is described, in which the DNA of cells in S phase are labeled with two different probes. This method makes it possible to study the relationship between DNA synthesis and the uptake and/or incorporation of chemotherapeutic agents into normal or neoplastic cells. An example is provided in which the incorporation of /sup 3/H-cytarabine into DNA is demonstrated to occur only in cells which were synthesizing DNA during exposure to /sup 3/H-cytarabine. Other radioactively labeled probes can be used as well.

Cytotoxicity and mitogenicity assays with real-time and label-free monitoring of human granulosa cells with an impedance-based signal processing technology intergrating micro-electronics and cell biology.

PubMed

Oktem, Ozgur; Bildik, Gamze; Senbabaoglu, Filiz; Lack, Nathan A; Akin, Nazli; Yakar, Feridun; Urman, Defne; Guzel, Yilmaz; Balaban, Basak; Iwase, Akira; Urman, Bulent

2016-04-01

A recently developed technology (xCelligence) integrating micro-electronics and cell biology allows real-time, uninterrupted and quantitative analysis of cell proliferation, viability and cytotoxicity by measuring the electrical impedance of the cell population in the wells without using any labeling agent. In this study we investigated if this system is a suitable model to analyze the effects of mitogenic (FSH) and cytotoxic (chemotherapy) agents with different toxicity profiles on human granulosa cells in comparison to conventional methods of assessing cell viability, DNA damage, apoptosis and steroidogenesis. The system generated the real-time growth curves of the cells, and determined their doubling times, mean cell indices and generated dose-response curves after exposure to cytotoxic and mitogenic stimuli. It accurately predicted the gonadotoxicity of the drugs and distinguished less toxic agents (5-FU and paclitaxel) from more toxic ones (cisplatin and cyclophosphamide). This platform can be a useful tool for specific end-point assays in reproductive toxicology. Copyright © 2015 Elsevier Inc. All rights reserved.

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. © 2013 John Wiley & Sons A/S.

Magnetic Targeting Enhances Engraftment and Functional Benefit of Iron-Labeled Cardiosphere-Derived Cells in Myocardial Infarction

PubMed Central

Cheng, Ke; Li, Tao-Sheng; Malliaras, Konstantinos; Davis, Darryl; Zhang, Yiqiang; Marbán, Eduardo

2010-01-01

Rationale The success of cardiac stem cell therapies is limited by low cell retention, due at least in part to washout via coronary veins. Objective We sought to counter the efflux of transplanted cells by rendering them magnetically-responsive and imposing an external magnetic field on the heart during and immediately after injection. Methods and Results Cardiosphere-derived cells (CDCs) were labeled with superparamagnetic microspheres (SPMs). In vitro studies revealed that cell viability and function were minimally affected by SPM labeling. SPM-labeled rat CDCs were injected intramyocardially, with and without a superimposed magnet. With magnetic targeting, cells were visibly attracted towards the magnet and accumulated around the ischemic zone. In contrast, the majority of non-targeted cells washed out immediately after injection. Fluorescence imaging revealed more retention of transplanted cells in the heart, and less migration into other organs, in the magnetically-targeted group. Quantitative PCR confirmed that magnetic targeting enhanced cell retention (at 24 hours) and engraftment (at 3 weeks) in the recipient hearts by ∼3-fold compared to non-targeted cells. Morphometric analysis revealed maximal attenuation of LV remodeling, and echocardiography showed the greatest functional improvement, in the magnetic targeting group. Histologically, more engrafted cells were evident with magnetic targeting, but there was no incremental inflammation. Conclusion Magnetic targeting enhances cell retention, engraftment and functional benefit. This novel method to improve cell therapy outcomes offers the potential for rapid translation into clinical applications. PMID:20378859

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.

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

USDA-ARS?s Scientific Manuscript database

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

Emergence of cytotoxic resistance in cancer cell populations: Single-cell mechanisms and population-level consequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lorenzi, Tommaso; Chisholm, Rebecca H.; Lorz, Alexander

We formulate an individual-based model and a population model of phenotypic evolution, under cytotoxic drugs, in a cancer cell population structured by the expression levels of survival-potential and proliferation-potential. We apply these models to a recently studied experimental system. Our results suggest that mechanisms based on fundamental laws of biology can reversibly push an actively-proliferating, and drug-sensitive, cell population to transition into a weakly-proliferative and drug-tolerant state, which will eventually facilitate the emergence of more potent, proliferating and drug-tolerant cells.

Application of immunogold-silver staining and immunoenzymatic methods in multiple labelling of human pancreatic Langerhans islet cells.

PubMed

Krenács, T; Lászik, Z; Dobó, E

1989-01-01

The use of immunogold-silver staining (IGSS) combined with immunoperoxidase and/or immunoalkaline phosphatase methods for the simultaneous demonstration of pancreatic islet cell hormones on routinely fixed paraffin-embedded human tissue sections was examined. If IGSS was applied first, the black colour of silver-enhanced colloidal gold on doubly immunostained sections contrasted with the colours of most of the chromogens used generally in the 2 immunoenzymatic methods. If IGSS was followed by immunoalkaline phosphatase and immunoperoxidase techniques in optional sequence, 3 different hormone-containing cell types could be stained simultaneously without non-specific cross-reactions. IGSS and immunoalkaline phosphatase methods, together with 2 kinds of non-cross-reacting immunoperoxidase systems, permitted the detection of 4 distinct antigens on the same tissue section. Multiple immunohistochemical labelling of the endocrine pancreas provides an opportunity for the correct and rapid analysis of the topographic and morphometric relationships between different hormone-producing cell populations under both normal and pathological conditions. IGSS is of great potential for the simultaneous immunolabelling of antigens situated within separate cells.

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

NASA Astrophysics Data System (ADS)

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

2016-07-01

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

Chemotaxis of cancer cells in three-dimensional environment monitored label-free by quantitative phase digital holographic microscopy

NASA Astrophysics Data System (ADS)

Kemper, Björn; Schnekenburger, Jürgen; Ketelhut, Steffi

2017-02-01

We investigated the capabilities of digital holographic microscopy (DHM) for label-free quantification of the response of living single cells to chemical stimuli in 3D assays. Fibro sarcoma cells were observed in a collagen matrix inside 3D chemotaxis chambers with a Mach-Zehnder interferometer-based DHM setup. From the obtained series of quantitative phase images, the migration trajectories of single cells were retrieved by automated cell tracking and subsequently analyzed for maximum migration distance and motility. Our results demonstrate DHM as a highly reliable and efficient tool for label-free quantification of chemotaxis in 2D and 3D environments.

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

PubMed Central

Patel, Chetan D; Agarwal, Snehlata; Seth, Sandeep; Mohanty, Sujata; Aggarwal, Himesh; Gupta, Namit

2014-01-01

Bone marrow stem cells having myogenic potential are promising candidates for various cell-based therapies for myocardial disease. We present here images showing homing of technetium-99m (Tc-99m) hexamethylpropyleneamine oxime (HMPAO) labeled stem cells in the infarcted myocardium from a pilot study conducted to radio-label part of the stem cells in patients enrolled in a stem cell clinical trial for recent myocardial infarction. PMID:25400375

An optimized method for measuring fatty acids and cholesterol in stable isotope-labeled cells

PubMed Central

Argus, Joseph P.; Yu, Amy K.; Wang, Eric S.; Williams, Kevin J.; Bensinger, Steven J.

2017-01-01

Stable isotope labeling has become an important methodology for determining lipid metabolic parameters of normal and neoplastic cells. Conventional methods for fatty acid and cholesterol analysis have one or more issues that limit their utility for in vitro stable isotope-labeling studies. To address this, we developed a method optimized for measuring both fatty acids and cholesterol from small numbers of stable isotope-labeled cultured cells. We demonstrate quantitative derivatization and extraction of fatty acids from a wide range of lipid classes using this approach. Importantly, cholesterol is also recovered, albeit at a modestly lower yield, affording the opportunity to quantitate both cholesterol and fatty acids from the same sample. Although we find that background contamination can interfere with quantitation of certain fatty acids in low amounts of starting material, our data indicate that this optimized method can be used to accurately measure mass isotopomer distributions for cholesterol and many fatty acids isolated from small numbers of cultured cells. Application of this method will facilitate acquisition of lipid parameters required for quantifying flux and provide a better understanding of how lipid metabolism influences cellular function. PMID:27974366

Comparison of tumor biology of two distinct cell sub-populations in lung cancer stem cells.

PubMed

Wang, Jianyu; Sun, Zhiwei; Liu, Yongli; Kong, Liangsheng; Zhou, Shixia; Tang, Junlin; Xing, Hongmei Rosie

2017-11-14

Characterization of the stem-like properties of cancer stem cells (CSCs) remain indirect and qualitative, especially the ability of CSCs to undergo asymmetric cell division for self renewal and differentiation, a unique property of cells of stem origin. It is partly due to the lack of stable cellular models of CSCs. In this study, we developed a new approach for CSC isolation and purification to derive a CSC-enriched cell line (LLC-SE). By conducting five consecutive rounds of single cell cloning using the LLC-SE cell line, we obtained two distinct sub-population of cells within the Lewis lung cancer CSCs that employed largely symmetric division for self-renewal (LLC-SD) or underwent asymmetric division for differentiation (LLC-ASD). LLC-SD and LLC-ASD cell lines could be stably passaged in culture and be distinguished by cell morphology, stem cell marker, spheroid formation and subcutaneous tumor initiation efficiency, as well as orthotopic lung tumor growth, progression and survival. The ability LLC-ASD cells to undergo asymmetric division was visualized and quantified by the asymmetric segregation of labeled BrdU and NUMB to one of the two daughter cells in anaphase cell division. The more stem-like LLC-SD cells exhibited higher capacity for tumorigenesis and progression and shorter survival. As few as 10 LLC-SD could initiate subcutaneous tumor growth when transplanted to the athymic mice. Collectively, these observations suggest that the SD-type of cells appear to be on the top of the hierarchical order of the CSCs. Furthermore, they have lead to generated cellular models of CSC self-renewal for future mechanistic investigations.

Optimized labeling of bone marrow mesenchymal cells with superparamagnetic iron oxide nanoparticles and in vivo visualization by magnetic resonance imaging

PubMed Central

2011-01-01

Background Stem cell therapy has emerged as a promising addition to traditional treatments for a number of diseases. However, harnessing the therapeutic potential of stem cells requires an understanding of their fate in vivo. Non-invasive cell tracking can provide knowledge about mechanisms responsible for functional improvement of host tissue. Superparamagnetic iron oxide nanoparticles (SPIONs) have been used to label and visualize various cell types with magnetic resonance imaging (MRI). In this study we performed experiments designed to investigate the biological properties, including proliferation, viability and differentiation capacity of mesenchymal cells (MSCs) labeled with clinically approved SPIONs. Results Rat and mouse MSCs were isolated, cultured, and incubated with dextran-covered SPIONs (ferumoxide) alone or with poly-L-lysine (PLL) or protamine chlorhydrate for 4 or 24 hrs. Labeling efficiency was evaluated by dextran immunocytochemistry and MRI. Cell proliferation and viability were evaluated in vitro with Ki67 immunocytochemistry and live/dead assays. Ferumoxide-labeled MSCs could be induced to differentiate to adipocytes, osteocytes and chondrocytes. We analyzed ferumoxide retention in MSCs with or without mitomycin C pretreatment. Approximately 95% MSCs were labeled when incubated with ferumoxide for 4 or 24 hrs in the presence of PLL or protamine, whereas labeling of MSCs incubated with ferumoxide alone was poor. Proliferative capacity was maintained in MSCs incubated with ferumoxide and PLL for 4 hrs, however, after 24 hrs it was reduced. MSCs incubated with ferumoxide and protamine were efficiently visualized by MRI; they maintained proliferation and viability for up to 7 days and remained competent to differentiate. After 21 days MSCs pretreated with mitomycin C still showed a large number of ferumoxide-labeled cells. Conclusions The efficient and long lasting uptake and retention of SPIONs by MSCs using a protocol employing ferumoxide and

[18F]FDG labeling of neural stem cells for in vivo cell tracking with positron emission tomography: inhibition of tracer release by phloretin.

PubMed

Stojanov, Katica; de Vries, Erik F J; Hoekstra, Dick; van Waarde, Aren; Dierckx, Rudi A J O; Zuhorn, Inge S

2012-02-01

The introduction of neural stem cells into the brain has promising therapeutic potential for the treatment of neurodegenerative diseases. To monitor the cellular replacement therapy, that is, to determine stem cell migration, survival, and differentiation, in vivo tracking methods are needed. Ideally, these tracking methods are noninvasive. Noninvasive tracking methods that have been successfully used for the visualization of blood-derived progenitor cells include magnetic resonance imaging and radionuclide imaging using single-photon emission computed tomography (SPECT) and positron emission tomography (PET). The SPECT tracer In-111-oxine is suitable for stem cell labeling, but for studies in small animals, the higher sensitivity and facile quantification that can be obtained with PET are preferred. Here the potential of 2'-[18F]fluoro-2'-deoxy-D-glucose ([18F]-FDG), a PET tracer, for tracking of neural stem cell (NSCs) trafficking toward an inflammation site was investigated. [18F]-FDG turns out to be a poor radiopharmaceutical to label NSCs owing to the low labeling efficiency and substantial release of radioactivity from these cells. Efflux of [18F]-FDG from NSCs can be effectively reduced by phloretin in vitro, but inhibition of tracer release is insufficient in vivo for accurate monitoring of stem cell trafficking.

Readability of prescription labels and medication recall in a population of tertiary referral glaucoma patients.

PubMed

O'Hare, Fleur; Jeganathan, V Swetha E; Rokahr, Catherine G; Rogers, Sophie L; Crowston, Jonathan G

2009-12-01

To evaluate readability of eye drop labels and accurate recall of prescription instructions in a glaucoma population. A hospital-based, cross-sectional study. A trained, interviewer examined patient ability to read standard and larger font medication labels. A questionnaire was administered to ascertain accurate recall of prescribed eye drops. Clinical information was obtained through independent chart review. Glaucoma severity was classified according to a glaucoma staging system. The setting for the study was the glaucoma outpatient clinic, Royal Victorian Eye and Ear Hospital (Melbourne, Australia), a major tertiary referral centre. A total of 200 glaucoma patients (96.2% response), aged 45-90 years, on eye drops took part in the study. Non-English-speaking patients were excluded. The main outcome measure was the ability to read prescribed medication labels and accurately recall treatment regime was compared with glaucoma severity and the number of eye drops. Of the glaucoma patients, 12% were unable to read standard pharmacy labels. Only 5.5% were unable to read the larger font labels. Of the patients, 32% were not able to accurately recall the type of drops or prescribed frequency of instillation. An inability to read standard labels was associated with a threefold reduction in the likelihood of accurate medication recall (95% confidence intervals, 1.40-7.66, P < 0.05). Patients with three or more types of eye drops were five times less likely to recall their medications (95% confidence interval, 0.07-0.57, P < 0.05). Inability to read or recall prescribed eye drops was associated with glaucoma severity and the number of prescribed eye drops. These factors may impact significantly on patients' adherence to glaucoma medications.

Labeling adipose derived stem cell sheet by ultrasmall super-paramagnetic Fe3O4 nanoparticles and magnetic resonance tracking in vivo.

PubMed

Zhou, Shukui; Yin, Ting; Zou, Qingsong; Zhang, Kaile; Gao, Guo; Shapter, Joseph G; Huang, Peng; Fu, Qiang

2017-02-21

Cell sheet therapy has emerged as a potential therapeutic option for reparation and reconstruction of damaged tissues and organs. However, an effective means to assess the fate and distribution of transplanted cell sheets in a serial and noninvasive manner is still lacking. To investigate the feasibility of tracking Adipose derived stem cells (ADSCs) sheet in vivo using ultrasmall super-paramagnetic Fe 3 O 4 nanoparticles (USPIO), canine ADSCs were cultured and incubated with USPIO and 0.75 μg/ml Poly-L-Lysine (PLL) for 12 h. Labeling efficiency, cell viability, apoptotic cell rate were assessed to screen the optimum concentrations of USPIO for best labeling ADSCs. The results showed ADSCs were labeled by USPIO at an iron dose of 50 μg/ml for a 12 h incubation time, which can most efficiently mark cells and did not impair the cell survival, self-renewal, and proliferation capacity. USPIO-labeled ADSCs sheets can be easily and clearly detected in vivo and have persisted for at least 12 weeks. Our experiment confirmed USPIO was feasible for in vivo labeling of the ADSCs sheets with the optimal concentration of 50 μg Fe/ml and the tracing time is no less than 12 weeks.

3D imaging of cells in scaffolds: direct labelling for micro CT.

PubMed

Shepherd, David V; Shepherd, Jennifer H; Best, Serena M; Cameron, Ruth E

2018-06-12

The development of in-vitro techniques to characterise the behaviour of cells in biomedical scaffolds is a rapidly developing field. However, until now it has not been possible to visualise, directly in 3D, the extent of cell migration using a desktop X-ray microCT. This paper describes a new technique based on cell labelling with a radio opacifier (barium sulphate), which permits cell tracking without the need for destructive sample preparation. The ability to track cells is highlighted via a comparison of cell migration through demonstrator lyophilised collagen scaffolds with contrasting pore size and interconnectivity. The results demonstrate the ease with which the technique can be used to characterise the effects of scaffold architecture on cell infiltration.

Application of phasor plot and autofluorescence correction for study of heterogeneous cell population

PubMed Central

Szmacinski, Henryk; Toshchakov, Vladimir; Lakowicz, Joseph R.

2014-01-01

Abstract. Protein-protein interactions in cells are often studied using fluorescence resonance energy transfer (FRET) phenomenon by fluorescence lifetime imaging microscopy (FLIM). Here, we demonstrate approaches to the quantitative analysis of FRET in cell population in a case complicated by a highly heterogeneous donor expression, multiexponential donor lifetime, large contribution of cell autofluorescence, and significant presence of unquenched donor molecules that do not interact with the acceptor due to low affinity of donor-acceptor binding. We applied a multifrequency phasor plot to visualize FRET FLIM data, developed a method for lifetime background correction, and performed a detailed time-resolved analysis using a biexponential model. These approaches were applied to study the interaction between the Toll Interleukin-1 receptor (TIR) domain of Toll-like receptor 4 (TLR4) and the decoy peptide 4BB. TLR4 was fused to Cerulean fluorescent protein (Cer) and 4BB peptide was labeled with Bodipy TMRX (BTX). Phasor displays for multifrequency FLIM data are presented. The analytical procedure for lifetime background correction is described and the effect of correction on FLIM data is demonstrated. The absolute FRET efficiency was determined based on the phasor plot display and multifrequency FLIM data analysis. The binding affinity between TLR4-Cer (donor) and decoy peptide 4BB-BTX (acceptor) was estimated in a heterogeneous HeLa cell population. PMID:24770662

Chemical modification and labeling of glutamate residues at the stilbenedisulfonate site of human red blood cell band 3 protein.

PubMed

Jennings, M L; Anderson, M P

1987-02-05

A new method has been developed for the chemical modification and labeling of carboxyl groups in proteins. Carboxyl groups are activated with Woodward's reagent K (N-ethyl-5-phenylisoxazolium 3'-sulfonate), and the adducts are reduced with [3H]BH4. The method has been applied to the anion transport protein of the human red blood cell (band 3). Woodward's reagent K is a reasonably potent inhibitor of band 3-mediated anion transport; a 5-min exposure of intact cells to 2 mM reagent at pH 6.5 produces 80% inhibition of transport. The inhibition is a consequence of modification of residues that can be protected by 4,4'-dinitrostilbene-2,2'-disulfonate. Treatment of intact cells with Woodward's reagent K followed by B3H4 causes extensive labeling of band 3, with minimal labeling of intracellular proteins such as spectrin. Proteolytic digestion of the labeled protein reveals that both the 60- and the 35-kDa chymotryptic fragments are labeled and that the labeling of each is inhibitable by stilbenedisulfonate. If the reduction is performed at neutral pH the major labeled product is the primary alcohol corresponding to the original carboxylic acid. Liquid chromatography of acid hydrolysates of labeled affinity-purified band 3 shows that glutamate but not aspartate residues have been converted into the hydroxyl derivative. This is the first demonstration of the conversion of a glutamate carboxyl group to an alcohol in a protein. The labeling experiments reveal that there are two glutamate residues that are sufficiently close to the stilbenedisulfonate site for their labeling to be blocked by 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate and 4,4'-dinitrostilbene-2,2'-disulfonate.

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

NASA Astrophysics Data System (ADS)

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

2016-07-01

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

Label-Free Detection of Bacillus anthracis Spore Uptake in Macrophage Cells Using Analytical Optical Force Measurements.

PubMed

Hebert, Colin G; Hart, Sean; Leski, Tomasz A; Terray, Alex; Lu, Qin

2017-10-03

Understanding the interaction between macrophage cells and Bacillus anthracis spores is of significant importance with respect to both anthrax disease progression, spore detection for biodefense, as well as understanding cell clearance in general. While most detection systems rely on specific molecules, such as nucleic acids or proteins and fluorescent labels to identify the target(s) of interest, label-free methods probe changes in intrinsic properties, such as size, refractive index, and morphology, for correlation with a particular biological event. Optical chromatography is a label free technique that uses the balance between optical and fluidic drag forces within a microfluidic channel to determine the optical force on cells or particles. Here we show an increase in the optical force experienced by RAW264.7 macrophage cells upon the uptake of both microparticles and B. anthracis Sterne 34F2 spores. In the case of spores, the exposure was detected in as little as 1 h without the use of antibodies or fluorescent labels of any kind. An increase in the optical force was also seen in macrophage cells treated with cytochalasin D, both with and without a subsequent exposure to spores, indicating that a portion of the increase in the optical force arises independent of phagocytosis. These results demonstrate the capability of optical chromatography to detect subtle biological differences in a rapid and sensitive manner and suggest future potential in a range of applications, including the detection of biological threat agents for biodefense and pathogens for the prevention of sepsis and other diseases.

Fluorophore labeling of a cell-penetrating peptide induces differential effects on its cellular distribution and affects cell viability.

PubMed

Birch, Ditlev; Christensen, Malene Vinther; Staerk, Dan; Franzyk, Henrik; Nielsen, Hanne Mørck

2017-12-01

Cell-penetrating peptides constitute efficient delivery vectors, and studies of their uptake and mechanism of translocation typically involve fluorophore-labeled conjugates. In the present study, the influence of a number of specific fluorophores on the physico-chemical properties and uptake-related characteristics of penetratin were studied. An array of seven fluorophores belonging to distinct structural classes was examined, and the impact of fluorophore labeling on intracellular distribution and cytotoxicity was correlated to the physico-chemical properties of the conjugates. Exposure of several mammalian cell types to fluorophore-penetratin conjugates revealed a strong structure-dependent reduction in viability (1.5- to 20-fold lower IC 50 values as compared to those of non-labeled penetratin). Also, the degree of less severe effects on membrane integrity, as well as intracellular distribution patterns differed among the conjugates. Overall, neutral hydrophobic fluorophores or negatively charged fluorophores conferred less cytotoxicity as compared to the effect exerted by positively charged, hydrophobic fluorophores. The latter conjugates, however, exhibited less membrane association and more clearly defined intracellular distribution patterns. Thus, selection of the appropriate flurophore is critical. Copyright © 2017 Elsevier B.V. All rights reserved.

A non-genetic approach to labelling acute myeloid leukemia and bone marrow cells with quantum dots.

PubMed

Zheng, Yanwen; Tan, Dongming; Chen, Zheng; Hu, Chenxi; Mao, Zhengwei J; Singleton, Timothy P; Zeng, Yan; Shao, Xuejun; Yin, Bin

2014-06-01

The difficulty in manipulation of leukemia cells has long hindered the dissection of leukemia pathogenesis. We have introduced a non-genetic approach of marking blood cells, using quantum dots. We compared quantum dots complexed with different vehicles, including a peptide Tat, cationic polymer Turbofect and liposome. Quantum dots-Tat showed the highest efficiency of marking hematopoietic cells among the three vehicles. Quantum dots-Tat could also label a panel of leukemia cell lines at varied efficiencies. More uniform intracellular distributions of quantum dots in mouse bone marrow and leukemia cells were obtained with quantum dots-Tat, compared with the granule-like formation obtained with quantum dots-liposome. Our results suggest that quantum dots have provided a photostable and non-genetic approach that labels normal and malignant hematopoietic cells, in a cell type-, vehicle-, and quantum dot concentration-dependent manner. We expect for potential applications of quantum dots as an easy and fast marking tool assisting investigations of various types of blood cells in the future.

Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue

PubMed Central

Robertson, David; Savage, Kay; Reis-Filho, Jorge S; Isacke, Clare M

2008-01-01

Background Investigating the expression of candidate genes in tissue samples usually involves either immunohistochemical labelling of formalin-fixed paraffin-embedded (FFPE) sections or immunofluorescence labelling of cryosections. Although both of these methods provide essential data, both have important limitations as research tools. Consequently, there is a demand in the research community to be able to perform routine, high quality immunofluorescence labelling of FFPE tissues. Results We present here a robust optimised method for high resolution immunofluorescence labelling of FFPE tissues, which involves the combination of antigen retrieval, indirect immunofluorescence and confocal laser scanning microscopy. We demonstrate the utility of this method with examples of immunofluorescence labelling of human kidney, human breast and a tissue microarray of invasive human breast cancers. Finally, we demonstrate that stained slides can be stored in the short term at 4°C or in the longer term at -20°C prior to images being collected. This approach has the potential to unlock a large in vivo database for immunofluorescence investigations and has the major advantages over immunohistochemistry in that it provides higher resolution imaging of antigen localization and the ability to label multiple antigens simultaneously. Conclusion This method provides a link between the cell biology and pathology communities. For the cell biologist, it will enable them to utilise the vast archive of pathology specimens to advance their in vitro data into in vivo samples, in particular archival material and tissue microarrays. For the pathologist, it will enable them to utilise multiple antibodies on a single section to characterise particular cell populations or to test multiple biomarkers in limited samples and define with greater accuracy cellular heterogeneity in tissue samples. PMID:18366689

Comparison of pre-processing techniques for fluorescence microscopy images of cells labeled for actin.

PubMed

Muralidhar, Gautam S; Channappayya, Sumohana S; Slater, John H; Blinka, Ellen M; Bovik, Alan C; Frey, Wolfgang; Markey, Mia K

2008-11-06

Automated analysis of fluorescence microscopy images of endothelial cells labeled for actin is important for quantifying changes in the actin cytoskeleton. The current manual approach is laborious and inefficient. The goal of our work is to develop automated image analysis methods, thereby increasing cell analysis throughput. In this study, we present preliminary results on comparing different algorithms for cell segmentation and image denoising.

Phytoplankton IF-FISH: Species-specific labeling of cellular proteins by immunofluorescence (IF) with simultaneous species identification by fluorescence immunohybridization (FISH).

PubMed

Meek, Megan E; Van Dolah, Frances M

2016-05-01

Phytoplankton rarely occur as unialgal populations. Therefore, to study species-specific protein expression, indicative of physiological status in natural populations, methods are needed that will both assay for a protein of interest and identify the species expressing it. Here we describe a protocol for IF-FISH, a dual labeling procedure using immunofluorescence (IF) labeling of a protein of interest followed by fluorescence in situ hybridization (FISH) to identify the species expressing that protein. The protocol was developed to monitor expression of the cell cycle marker proliferating cell nuclear antigen (PCNA) in the red tide dinoflagellate, Karenia brevis, using a large subunit (LSU) rRNA probe to identify K. brevis in a mixed population of morphologically similar Karenia species. We present this protocol as proof of concept that IF-FISH can be successfully applied to phytoplankton cells. This method is widely applicable for the analysis of single-cell protein expression of any protein of interest within phytoplankton communities. Copyright © 2016 Elsevier B.V. All rights reserved.

Separable Bilayer Microfiltration Device for Label-Free Enrichment of Viable Circulating Tumor Cells.

PubMed

Hao, Sijie; Nisic, Merisa; He, Hongzhang; Tai, Yu-Chong; Zheng, Si-Yang

2017-01-01

Analysis of rare circulating tumor cells enriched from metastatic cancer patients yields critical information on disease progression, therapy response, and the mechanism of cancer metastasis. Here we describe in detail a label-free enrichment process of circulating tumor cells based on its unique physical properties (size and deformability). Viable circulating tumor cells can be successfully enriched and analyzed, or easily released for further characterization due to the novel separable two-layer design.

A simple method for purification of vestibular hair cells and non-sensory cells, and application for proteomic analysis.

PubMed

Herget, Meike; Scheibinger, Mirko; Guo, Zhaohua; Jan, Taha A; Adams, Christopher M; Cheng, Alan G; Heller, Stefan

2013-01-01

Mechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level. Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.

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. © 2015 International Society for Advancement of Cytometry.

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.

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

NHS-based Tandem Mass Tagging of Proteins at the Level of Whole Cells: A Critical Evaluation in Comparison to Conventional TMT-Labeling Approaches for Quantitative Proteome Analysis.

PubMed

Megger, Dominik A; Pott, Leona L; Rosowski, Kristin; Zülch, Birgit; Tautges, Stephanie; Bracht, Thilo; Sitek, Barbara

2017-01-01

Tandem mass tags (TMT) are usually introduced at the levels of isolated proteins or peptides. Here, for the first time, we report the labeling of whole cells and a critical evaluation of its performance in comparison to conventional labeling approaches. The obtained results indicated that TMT protein labeling using intact cells is generally possible, if it is coupled to a subsequent enrichment using anti-TMT antibody. The quantitative results were similar to those obtained after labeling of isolated proteins and both were found to be slightly complementary to peptide labeling. Furthermore, when using NHS-based TMT, no specificity towards cell surface proteins was observed in the case of cell labeling. In summary, the conducted study revealed first evidence for the general possibility of TMT cell labeling and highlighted limitations of NHS-based labeling reagents. Future studies should therefore focus on the synthesis and investigation of membrane impermeable TMTs to increase specificity towards cell surface proteins.

Collective dynamics of cell migration and cell rearrangements

NASA Astrophysics Data System (ADS)

Kabla, Alexandre

Understanding multicellular processes such as embryo development or cancer metastasis requires to decipher the contributions of local cell autonomous behaviours and long range interactions with the tissue environment. A key question in this context concerns the emergence of large scale coordination in cell behaviours, a requirement for collective cell migration or convergent extension. I will present a few examples where physical and mechanical aspects play a significant role in driving tissue scale dynamics.