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Sample records for activated cell sorting

  1. Fluorescence activated cell sorting of plant protoplasts.

    Science.gov (United States)

    Bargmann, Bastiaan O R; Birnbaum, Kenneth D

    2010-02-18

    High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis. An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: P(SCR;)::GFP (endodermis and quiescent center) and P(WOX5;)::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution. FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time. This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce

  2. Pattern matching based active optical sorting of colloids/cells

    Science.gov (United States)

    Verma, R. S.; Dasgupta, R.; Ahlawat, S.; Kumar, N.; Uppal, A.; Gupta, P. K.

    2013-08-01

    We report active optical sorting of colloids/cells by employing a cross correlation based pattern matching technique for selection of the desired objects and thereafter sorting using dynamically controllable holographic optical traps. The problem of possible collision between the different sets of objects during sorting was avoided by raising one set of particles to a different plane. We also present the results obtained on using this approach for some representative applications such as sorting of silica particles of two different sizes, of closely packed colloids and of white blood cells and red blood cells from a mixture of the two.

  3. Buoyancy-activated cell sorting using targeted biotinylated albumin microbubbles.

    Directory of Open Access Journals (Sweden)

    Yu-Ren Liou

    Full Text Available Cell analysis often requires the isolation of certain cell types. Various isolation methods have been applied to cell sorting, including fluorescence-activated cell sorting and magnetic-activated cell sorting. However, these conventional approaches involve exerting mechanical forces on the cells, thus risking cell damage. In this study we applied a novel isolation method called buoyancy-activated cell sorting, which involves using biotinylated albumin microbubbles (biotin-MBs conjugated with antibodies (i.e., targeted biotin-MBs. Albumin MBs are widely used as contrast agents in ultrasound imaging due to their good biocompatibility and stability. For conjugating antibodies, biotin is conjugated onto the albumin MB shell via covalent bonds and the biotinylated antibodies are conjugated using an avidin-biotin system. The albumin microbubbles had a mean diameter of 2 μm with a polydispersity index of 0.16. For cell separation, the MDA-MB-231 cells are incubated with the targeted biotin-MBs conjugated with anti-CD44 for 10 min, centrifuged at 10 g for 1 min, and then allowed 1 hour at 4 °C for separation. The results indicate that targeted biotin-MBs conjugated with anti-CD44 antibodies can be used to separate MDA-MB-231 breast cancer cells; more than 90% of the cells were collected in the MB layer when the ratio of the MBs to cells was higher than 70:1. Furthermore, we found that the separating efficiency was higher for targeted biotin-MBs than for targeted avidin-incorporated albumin MBs (avidin-MBs, which is the most common way to make targeted albumin MBs. We also demonstrated that the recovery rate of targeted biotin-MBs was up to 88% and the sorting purity was higher than 84% for a a heterogenous cell population containing MDA-MB-231 cells (CD44(+ and MDA-MB-453 cells (CD44-, which are classified as basal-like breast cancer cells and luminal breast cancer cells, respectively. Knowing that the CD44(+ is a commonly used cancer-stem-cell

  4. Towards high-throughput microfluidic Raman-activated cell sorting.

    Science.gov (United States)

    Zhang, Qiang; Zhang, Peiran; Gou, Honglei; Mou, Chunbo; Huang, Wei E; Yang, Menglong; Xu, Jian; Ma, Bo

    2015-09-21

    Raman-activated cell sorting (RACS) is a promising single-cell analysis technology that is able to identify and isolate individual cells of targeted type, state or environment from an isogenic population or complex consortium of cells, in a label-free and non-invasive manner. However, compared with those widely used yet labeling-required or staining-dependent cell sorting technologies such as FACS and MACS, the weak Raman signal greatly limits the further development of the existing RACS systems to achieve higher throughput. Strategies that can tackle this bottleneck include, first, improvement of Raman-acquisition efficiency and quality based on advanced Raman spectrometers and enhanced Raman techniques; second, development of novel microfluidic devices for cell sorting followed by integration into a complete RACS system. Exploiting these strategies, prototypes for a new generation of RACS have been demonstrated, such as flow-based OT-RACS, DEP-RACS, and SERS/CARS flow cytometry. Such high-throughput microfluidic RACS can provide biologists with a powerful single-cell analysis tool to explore the scientific questions or applications that have been beyond the reach of FACS and MACS.

  5. Cell sorting in development.

    Science.gov (United States)

    Krens, S F Gabby; Heisenberg, Carl-Philipp

    2011-01-01

    During the development of multicellular organisms, cell fate specification is followed by the sorting of different cell types into distinct domains from where the different tissues and organs are formed. Cell sorting involves both the segregation of a mixed population of cells with different fates and properties into distinct domains, and the active maintenance of their segregated state. Because of its biological importance and apparent resemblance to fluid segregation in physics, cell sorting was extensively studied by both biologists and physicists over the last decades. Different theories were developed that try to explain cell sorting on the basis of the physical properties of the constituent cells. However, only recently the molecular and cellular mechanisms that control the physical properties driving cell sorting, have begun to be unraveled. In this review, we will provide an overview of different cell-sorting processes in development and discuss how these processes can be explained by the different sorting theories, and how these theories in turn can be connected to the molecular and cellular mechanisms driving these processes.

  6. Magnetic activated cell sorting (MACS): utility in assisted reproduction.

    Science.gov (United States)

    Makker, Kartikeya; Agarwal, Ashok; Sharma, Rakesh K

    2008-07-01

    Assisted reproductive techniques (ART) have now been extensively incorporated in the management of infertile couples. But even after rapid methodological and technological advances the success rates of these procedures have been below expectations. This has led to development of many sperm preparation protocols to obtain an ideal semen sample for artificial reproduction. Sperm apoptosis has been heavily linked to failures in reproductive techniques. One of the earliest changes shown by apoptotic spermatozoa is externalization of phosphatidyl serine. Magnetic activated cell sorting (MACS) is a novel sperm preparation technique that separates apoptotic and non-apoptotic spermatozoa based on the expression of phosphatidylserine. This has led to the incorporation of MACS as a sperm preparation technique. The review highlights the principle and mechanism of this novel technique and enumerates its advantages as a sperm preparation technique. Its utility in ART as an efficient tool for sperm recovery and its application in cryopreservation of semen samples is also explained.

  7. Fluorescence-Activated Cell Sorting Analysis of Heterotypic Cell-in-Cell Structures.

    Science.gov (United States)

    He, Meifang; Huang, Hongyan; Wang, Manna; Chen, Ang; Ning, Xiangkai; Yu, Kaitao; Li, Qihong; Li, Wen; Ma, Li; Chen, Zhaolie; Wang, Xiaoning; Sun, Qiang

    2015-04-27

    Cell-in-cell structures (CICs), characterized by the presence of one or more viable cells inside another one, were recently found important player in development, immune homeostasis and tumorigenesis etc. Incompatible with ever-increasing interests on this unique phenomenon, reliable methods available for high throughput quantification and systemic investigation are lacking. Here, we report a flow cytometry-based method for rapid analysis and sorting of heterotypic CICs formed between lymphocytes and tumor cells. In this method, cells were labeled with fluorescent dyes for fluorescence-activated cell sorting (FACS) by flow cytometry, conditions for reducing cell doublets were optimized such that high purity (>95%) of CICs could be achieved. By taking advantage of this method, we analyzed CICs formation between different cell pairs, and found that factors from both internalized effector cells and engulfing target cells affect heterotypic CICs formation. Thus, flow cytometry-based FACS analysis would serve as a high throughput method to promote systemic researches on CICs.

  8. Fluorescence-Activated Cell Sorting of Live Versus Dead Bacterial Cells and Spores

    Science.gov (United States)

    Bernardini, James N.; LaDuc, Myron T.; Diamond, Rochelle; Verceles, Josh

    2012-01-01

    This innovation is a coupled fluorescence-activated cell sorting (FACS) and fluorescent staining technology for purifying (removing cells from sampling matrices), separating (based on size, density, morphology, and live versus dead), and concentrating cells (spores, prokaryotic, eukaryotic) from an environmental sample.

  9. Individual cell sorting.

    Science.gov (United States)

    Stovel, R T; Sweet, R G

    1979-01-01

    Current cell sorting machines do not preserve the individual identity of processed cells; after analysis, the cells are assigned to a subpopulation where they are pooled with other similar cells. This paper reports progress on a system that sorts cells individually to precise locations on a microscope slide and preserves them for further observation with a light microscope while recording flow measurement data for each cell. Various electronic and mechanical modifications to an existing sorting machine are described that increase drop placement accuracy and permit individual cell sorting.

  10. PCR-activated cell sorting for cultivation-free enrichment and sequencing of rare microbes.

    Directory of Open Access Journals (Sweden)

    Shaun W Lim

    Full Text Available Microbial systems often exhibit staggering diversity, making the study of rare, interesting species challenging. For example, metagenomic analyses of mixed-cell populations are often dominated by the sequences of the most abundant organisms, while those of rare microbes are detected only at low levels, if at all. To overcome this, selective cultivation or fluorescence-activated cell sorting (FACS can be used to enrich for the target species prior to sequence analysis; however, since most microbes cannot be grown in the lab, cultivation strategies often fail, while cell sorting requires techniques to uniquely label the cell type of interest, which is often not possible with uncultivable microbes. Here, we introduce a culture-independent strategy for sorting microbial cells based on genomic content, which we term PCR-activated cell sorting (PACS. This technology, which utilizes the power of droplet-based microfluidics, is similar to FACS in that it uses a fluorescent signal to uniquely identify and sort target species. However, PACS differs importantly from FACS in that the signal is generated by performing PCR assays on the cells in microfluidic droplets, allowing target cells to be identified with high specificity with suitable design of PCR primers and TaqMan probes. The PACS assay is general, requires minimal optimization and, unlike antibody methods, can be developed without access to microbial antigens. Compared to non-specific methods in which cells are sorted based on size, granularity, or the ability to take up dye, PACS enables genetic sequence-specific sorting and recovery of the cell genomes. In addition to sorting microbes, PACS can be applied to eukaryotic cells, viruses, and naked nucleic acids.

  11. Fluorescence activated cell sorting via a focused traveling surface acoustic beam.

    Science.gov (United States)

    Ma, Zhichao; Zhou, Yinning; Collins, David J; Ai, Ye

    2017-09-12

    Fluorescence activated cell sorting (FACS) has become an essential technique widely exploited in biological studies and clinical applications. However, current FACS systems are quite complex, expensive, bulky, and pose potential sample contamination and biosafety issues due to the generation of aerosols in an open environment. Microfluidic technology capable of precise cell manipulation has great potential to reinvent and miniaturize conventional FACS systems. In this work, we demonstrate a benchtop scale FACS system that makes use of a highly focused traveling surface acoustic wave beam to sort out micron-sized particles and biological cells upon fluorescence interrogation at ∼kHz rates. The highly focused acoustic wave beam has a width of ∼50 μm that enables highly accurate sorting of individual particles and cells. We have applied our acoustic FACS system to isolate fluorescently labeled MCF-7 breast cancer cells from diluted whole blood samples with the purity of sorted MCF-7 cells higher than 86%. The cell viability before and after acoustic sorting is higher than 95%, indicating excellent biocompatibility that should enable a variety of cell sorting applications in biomedical research.

  12. Selection of nonapoptotic sperm by magnetic-activated cell sorting in Senegalese sole (Solea senegalensis).

    Science.gov (United States)

    Valcarce, D G; Herráez, M P; Chereguini, O; Rodríguez, C; Robles, V

    2016-09-15

    Senegalese sole (Solea senegalensis) is a promising species in aquaculture. However, owing to decreased sperm quality in F1 generations and the absence of courtship in those individuals born in captivity, artificial fertilization is being used to generate new progenies. The objective of this study was to implement a sperm selection method for nonapoptotic sperm subpopulation recovery before sperm cryopreservation. In particular, magnetic-activated cell sorting is used to eliminate apoptotic spermatozoa. This study represents the proof-of-concept for magnetic-activated cell sorting applicability in teleost species relevant in aquaculture. Apoptotic cell population was studied by flow cytometry using YO-PRO-1 and a caspase detection kit. Also, reactive oxygen species were measured in sperm samples. Our data demonstrated that caspase detection is more specific than YO-PRO-1 in the identification of apoptotic cells in S senegalensis seminal samples. The results showed that the percentage of apoptotic cells (caspase positive) was significantly higher (P = 0.04) in seminal samples from F1 than that from wild individuals. Magnetic-activated cell sorting removed a significant number of apoptotic cells from the samples (54% and 75% in wild and F1 individuals, respectively), decreasing the level of cells positive for reactive oxygen species (P = 0.17). In conclusion, this technique reduces the percentage of nonfunctional spermatozoa in a seminal sample before cryopreservation. This novel technique can be applied directly in the aquaculture industry.

  13. Fluorescent activated cell sorting: an effective approach to study dendritic cell subsets in human atherosclerotic plaques.

    Science.gov (United States)

    Van Brussel, Ilse; Ammi, Rachid; Rombouts, Miche; Cools, Nathalie; Vercauteren, Sven R; De Roover, Dominique; Hendriks, Jeroen M H; Lauwers, Patrick; Van Schil, Paul E; Schrijvers, Dorien M

    2015-02-01

    Different immune cell types are present within atherosclerotic plaques. Dendritic cells (DC) are of special interest, since they are considered as the 'center of the immuniverse'. Identifying inflammatory DC subtypes within plaques is important for a better understanding of the lesion pathogenesis and pinpoints their contribution to the atherosclerotic process. We have developed a flow cytometry-based method to characterize and isolate different DC subsets (i.e. CD11b(+), Clec9A(+) and CD16(+) conventional (c)DC and CD123(+) plasmacytoid (p)DC) in human atherosclerotic plaques. We revealed a predominance of pro-inflammatory CD11b(+) DC in advanced human lesions, whereas atheroprotective Clec9A(+) DC were almost absent. CD123(+) pDC and CD16(+) DC were also detectable in plaques. Remarkably, plaques from distinct anatomical locations exhibited different cellular compositions: femoral plaques contained less CD11b(+) and Clec9A(+) DC than carotid plaques. Twice as many monocytes/macrophages were observed compared to DC. Moreover, relative amounts of T cells/B cells/NK cells were 6 times as high as DC numbers. For the first time, fluorescent activated cell sorting analysis of DC subsets in human plaques indicated a predominance of CD11b(+) cDC, in comparison with other DC subsets. Isolation of the different subsets will facilitate detailed functional analysis and may have significant implications for tailoring appropriate therapy.

  14. Characterization of pancreatic stem cells derived from adult human pancreas ducts by fluorescence activated cell sorting

    Institute of Scientific and Technical Information of China (English)

    Han-Tso Lin; Shih-Hwa Chiou; Chung-Lan Kao; Yi-Ming Shyr; Chien-Jen Hsu; Yih-Wen Tarng; Larry L-T Ho; Ching-Fai Kwok; Hung-Hai Ku

    2006-01-01

    AIM: To isolate putative pancreatic stem cells (PSCs)from human adult tissues of pancreas duct using serumfree, conditioned medium. The characterization of surface phenotype of these PSCs was analyzed by flow cytometry. The potential for pancreatic lineage and the capability of β-cell differentiation in these PSCs were evaluated as well.METHODS: By using serum-free medium supplemented with essential growth factors, we attempted to isolate the putative PSCs which has been reported to express nestin and pdx-1. The MatrigelTM was employed to evaluate the differential capacity of isolated cells. Dithizone staining, insulin content/secretion measurement, and immunohistochemistry staining were used to monitor the differentiation. Fluorescence activated cell sorting (FACS)was used to detect the phenotypic markers of putative PSCs.RESULTS: A monolayer of spindle-like cells was cultivated. The putative PSCs expressed pdx-1 and nestin.They were also able to differentiate into insulin-, glucagon-, and somatostatin-positive cells. The spectrum of phenotypic markers in PSCs was investigated; a similarity was revealed when using human bone marrow-derived stem cells as the comparative experiment, such as CD29,CD44, CD49, CD50, CD51, CD62E, PDGFR-α, CD73 (SH2),CD81, CD105(SH3).CONCLUSION: In this study, we successfully isolated PSCs from adult human pancreatic duct by using serumfree medium. These PSCs not only expressed nestin and pdx-1 but also exhibited markers attributable to mesenchymal stem cells. Although work is needed to elucidate the role of these cells, the application of these PSCs might be therapeutic strategies for diabetes mellitus.

  15. The use of fluorescence-activated cell sorting in studying plant development and environmental responses.

    Science.gov (United States)

    Carter, Anthony D; Bonyadi, Roxanna; Gifford, Miriam L

    2013-01-01

    Fluorescence-Activated Cell Sorting (FACS) is a powerful tool that enables plant growth and development to be studied at the cellular level. Flow cytometry is used to isolate subpopulations of cells, such as those of specific cell types, or cells at particular developmental stages that have been marked with fluorescent proteins. Transgenic technology has given us the ability to generate plants that express fluorescent proteins, not just constitutively in particular cell types, but also dynamically in response to endogenous or external factors. By processing such transgenic lines with FACS, it is possible to isolate distinct populations of cells in a wide range of likely response states for further analysis. This is particularly useful for investigating biological mechanisms in plants because the control of growth and development is manifest at the cell type level. Furthermore, the specificity of the resulting data enables fine modelling of the transcriptional networks that exert systems-level control of the transcriptome; hence key regulators of responses and processes in the plant can be identified. In this review, the current state of the art for FACS methods in plants is explored by means of case studies of research in which cell sorting allowed us to make significant new discoveries.

  16. Separation of SSEA-4 and TRA-1-60 labelled undifferentiated human embryonic stem cells from a heterogeneous cell population using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS).

    Science.gov (United States)

    Fong, Chui Yee; Peh, Gary S L; Gauthaman, Kalamegam; Bongso, Ariff

    2009-03-01

    A major concern in human embryonic stem cell (hESC)-derived cell replacement therapy is the risk of tumorigenesis from undifferentiated hESCs residing in the population of hESC-derived cells. Separation of these undifferentiated hESCs from the differentiated derivatives using cell sorting methods may be a plausible approach in overcoming this problem. We therefore explored magnetic activated cell sorting (MACS) and fluorescence activated cell sorting (FACS) to separate labelled undifferentiated hESCs from a heterogeneous population of hESCs and hepatocellular carcinoma cells (HepG2) deliberately mixed respectively at different ratios (10:90, 20:80, 30:70, 40:60 and 50:50) to mimic a standard in vitro differentiation protocol, instead of using a hESC-differentiated cell population, so that we could be sure of the actual number of cells separated. HES-3 and HES-4 cells were labelled in separate experiments for the stem cell markers SSEA-4 and TRA-1-60 using primary antibodies. Anti-PE magnetic microbeads that recognize the PE-conjugated SSEA-4 labelled hESCs was added to the heterogeneous cell mixture and passed through the MACS column. The cells that passed through the column ('flow-through' fraction) and those retained ('labelled' fraction') were subsequently analysed using FACS. The maximum efficacy of hESCs retention using MACS was 81.0 +/- 2.9% (HES-3) and 83.6 +/- 4.2% (HES-4). Using FACS, all the undifferentiated hESCs labelled with the two cell-surface markers could be removed by selective gating. Both hESCs and HepG2 cells in the 'flow-through' fraction following MACS separation were viable in culture whereas by FACS separation only the HepG2 cells were viable. FACS efficiently helps to eliminate the undifferentiated hESCs based on their cell-surface antigens expressed.

  17. Genetic Screening for Bacterial Mutants in Liquid Growth Media By Fluorescence-Activated Cell Sorting

    Science.gov (United States)

    Abuaita, Basel H.; Withey, Jeffrey H.

    2010-01-01

    Many bacterial pathogens have defined in vitro virulence inducing conditions in liquid media which lead to production of virulence factors important during an infection. Identifying mutants that no longer respond to virulence inducing conditions will increase our understanding of bacterial pathogenesis. However, traditional genetic screens require growth on solid media. Bacteria in a single colony are in every phase of the growth curve, which complicates the analysis and make screens for growth phase-specific mutants problematic. Here, we utilize fluorescence-activated cell sorting in conjunction with random transposon mutagenesis to isolate bacteria grown in liquid media that are defective in virulence activation. This method permits analysis of an entire bacterial population in real time and selection of individual bacterial mutants with the desired gene expression profile at any time point after induction. We have used this method to identify Vibrio cholerae mutants defective in virulence induction. PMID:21094189

  18. Isolation of circulating tumor cells by immunomagnetic enrichment and fluorescence-activated cell sorting (IE/FACS) for molecular profiling.

    Science.gov (United States)

    Magbanua, Mark Jesus M; Park, John W

    2013-12-01

    Circulating tumor cells (CTCs) are cells shed by the primary tumor into the blood stream capable of initiating distant metastasis. In the past decade, numerous assays have been developed to reliably detect these extremely rare cells. However, methods for purification of CTCs with little or no contamination of normal blood cells for molecular profiling are limited. We have developed a novel protocol to isolate CTCs by combining immunomagnetic enrichment and fluorescence-activated cell sorting (IE/FACS). The two-part assay includes (1) immunomagnetic capture using magnetic beads conjugated to monoclonal antibody against an epithelial cell adhesion marker (EpCAM) to enrich for tumor cells; and (2) FACS analysis using EpCAM to purify tumor cells away from mononuclear cells of hematopoietic lineage. Downstream molecular analyses of single and pooled cells confirmed the isolation of highly pure CTCs with characteristics typical that of malignant cells.

  19. Rapid isolation of antibody from a synthetic human antibody library by repeated fluorescence-activated cell sorting (FACS.

    Directory of Open Access Journals (Sweden)

    Sung Sun Yim

    Full Text Available Antibodies and their derivatives are the most important agents in therapeutics and diagnostics. Even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. Here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (FACS. First, we constructed a library of synthetic human antibody in which single-chain variable fragment (scFv was expressed in the periplasm of Escherichia coli. After labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. After repeating this sorting, the positive clones were completely enriched in several hours. Thus, we screened the library against three viral antigens, including the H1N1 influenza virus, Hepatitis B virus, and Foot-and-mouth disease virus. Finally, the potential antibody candidates, which show K(D values between 10 and 100 nM against the target antigens, could be successfully isolated even though the library was relatively small (∼ 10(6. These results show that repeated FACS screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required.

  20. Efficient selective breeding of live oil-rich Euglena gracilis with fluorescence-activated cell sorting.

    Science.gov (United States)

    Yamada, Koji; Suzuki, Hideyuki; Takeuchi, Takuto; Kazama, Yusuke; Mitra, Sharbanee; Abe, Tomoko; Goda, Keisuke; Suzuki, Kengo; Iwata, Osamu

    2016-05-23

    Euglena gracilis, a microalgal species of unicellular flagellate protists, has attracted much attention in both the industrial and academic sectors due to recent advances in the mass cultivation of E. gracilis that have enabled the cost-effective production of nutritional food and cosmetic commodities. In addition, it is known to produce paramylon (β-1,3-glucan in a crystalline form) as reserve polysaccharide and convert it to wax ester in hypoxic and anaerobic conditions-a promising feedstock for biodiesel and aviation biofuel. However, there remain a number of technical challenges to be solved before it can be deployed in the competitive fuel market. Here we present a method for efficient selective breeding of live oil-rich E. gracilis with fluorescence-activated cell sorting (FACS). Specifically, the selective breeding method is a repetitive procedure for one-week heterotrophic cultivation, staining intracellular lipids with BODIPY(505/515), and FACS-based isolation of top 0.5% lipid-rich E. gracilis cells with high viability, after inducing mutation with Fe-ion irradiation to the wild type (WT). Consequently, we acquire a live, stable, lipid-rich E. gracilis mutant strain, named B1ZFeL, with 40% more lipid content on average than the WT. Our method paves the way for rapid, cost-effective, energy-efficient production of biofuel.

  1. Efficient selective breeding of live oil-rich Euglena gracilis with fluorescence-activated cell sorting

    Science.gov (United States)

    Yamada, Koji; Suzuki, Hideyuki; Takeuchi, Takuto; Kazama, Yusuke; Mitra, Sharbanee; Abe, Tomoko; Goda, Keisuke; Suzuki, Kengo; Iwata, Osamu

    2016-01-01

    Euglena gracilis, a microalgal species of unicellular flagellate protists, has attracted much attention in both the industrial and academic sectors due to recent advances in the mass cultivation of E. gracilis that have enabled the cost-effective production of nutritional food and cosmetic commodities. In addition, it is known to produce paramylon (β-1,3-glucan in a crystalline form) as reserve polysaccharide and convert it to wax ester in hypoxic and anaerobic conditions–a promising feedstock for biodiesel and aviation biofuel. However, there remain a number of technical challenges to be solved before it can be deployed in the competitive fuel market. Here we present a method for efficient selective breeding of live oil-rich E. gracilis with fluorescence-activated cell sorting (FACS). Specifically, the selective breeding method is a repetitive procedure for one-week heterotrophic cultivation, staining intracellular lipids with BODIPY505/515, and FACS-based isolation of top 0.5% lipid-rich E. gracilis cells with high viability, after inducing mutation with Fe-ion irradiation to the wild type (WT). Consequently, we acquire a live, stable, lipid-rich E. gracilis mutant strain, named B1ZFeL, with 40% more lipid content on average than the WT. Our method paves the way for rapid, cost-effective, energy-efficient production of biofuel. PMID:27212384

  2. A microfluidic device based on gravity and electric force driving for flow cytometry and fluorescence activated cell sorting.

    Science.gov (United States)

    Yao, Bo; Luo, Guo-an; Feng, Xue; Wang, Wei; Chen, Ling-xin; Wang, Yi-ming

    2004-12-01

    A novel method based on gravity and electric force driving of cells was developed for flow cytometry and fluorescence activated cell sorting in a microfluidic chip system. In the experiments cells flowed spontaneously under their own gravity in a upright microchip, passed through the detection region and then entered into the sorting electric field one by one at an average velocity of 0.55 mm s(-1) and were fluorescence activated cell sorted (FACS) by a switch-off activation program. In order to study the dynamical and kinematic characteristics of single cells in gravity and electric field of microchannels a physical and numerical module based on Newton's Law of motion was established and optimized. Hydroxylpropylmethyl cellulose (HPMC) was used to minimize cell assembling, sedimentation and adsorption to microchannels. This system was applied to estimate the necrotic and apoptotic effects of ultraviolet (UV) light on HeLa cells by exposing them to UV radiation for 10, 20 or 40 min and the results showed that UV radiation induced membrane damage contributed to the apoptosis and necrosis of HeLa cells.

  3. Sorting cells by their density

    Science.gov (United States)

    Norouzi, Nazila; Bhakta, Heran C.

    2017-01-01

    Sorting cells by their type is an important capability in biological research and medical diagnostics. However, most cell sorting techniques rely on labels or tags, which may have limited availability and specificity. Sorting different cell types by their different physical properties is an attractive alternative to labels because all cells intrinsically have these physical properties. But some physical properties, like cell size, vary significantly from cell to cell within a cell type; this makes it difficult to identify and sort cells based on their sizes alone. In this work we continuously sort different cells types by their density, a physical property with much lower cell-to-cell variation within a cell type (and therefore greater potential to discriminate different cell types) than other physical properties. We accomplish this using a 3D-printed microfluidic chip containing a horizontal flowing micron-scale density gradient. As cells flow through the chip, Earth’s gravity makes each cell move vertically to the point where the cell’s density matches the surrounding fluid’s density. When the horizontal channel then splits, cells with different densities are routed to different outlets. As a proof of concept, we use our density sorter chip to sort polymer microbeads by their material (polyethylene and polystyrene) and blood cells by their type (white blood cells and red blood cells). The chip enriches the fraction of white blood cells in a blood sample from 0.1% (in whole blood) to nearly 98% (in the output of the chip), a 1000x enrichment. Any researcher with access to a 3D printer can easily replicate our density sorter chip and use it in their own research using the design files provided as online Supporting Information. Additionally, researchers can simulate the performance of a density sorter chip in their own applications using the Python-based simulation software that accompanies this work. The simplicity, resolution, and throughput of this

  4. Endothelial cell high-enrichment from endovascular biopsy sample by laser capture microdissection and fluorescence activated cell sorting.

    Science.gov (United States)

    Sun, Zhengda; Su, Hua; Long, Brian; Sinclair, Elizabeth; Hetts, Steven W; Higashida, Randall T; Dowd, Christopher F; Halbach, Van V; Cooke, Daniel L

    2014-12-20

    Endovascular sampling and characterization from patients can provide very useful information about the pathogenesis of different vascular diseases, but it has been limited by the lack of an effective method of endothelial cell (EC) enrichment. We optimized the EC yield and enrichment from conventional guide wires by laser capture microdissection (LCM) and fluorescence activated cell sorting (FACS) technique, and addressed the feasibility of using these enriched ECs for downstream gene expression detection. Iliac artery endovascular samples from 10 patients undergoing routine catheter angiography were collected using conventional 0.038 in. J-shape guide wires. Each of these samples was equally divided into two parts, which were respectively used for EC enrichment by immunocytochemistry-coupled LCM or multiple color FACS. After RNA extraction and reverse transcription, the amplified cDNA was used for quantitative polymerase chain reaction (qPCR). Fixed ECs, with positive CD31 or vWF fluorescent signal and endothelial like nucleus, were successfully separated by LCM and live single ECs were sorted on FACS by a seven color staining panel. EC yields by LCM and FACS were 51 ± 22 and 149 ± 56 respectively (P < 0.001). The minimum number of fixed ECs from ICC-coupled LCM for acceptable qPCR results of endothelial marker genes was 30, while acceptable qPCR results as enriched by FACS were attainable from a single live EC. Both LCM and FACS can be used to enrich ECs from conventional guide wires and the enriched ECs can be used for downstream gene expression detection. FACS generated a higher EC yield and the sorted live ECs may be used for single cell gene expression detection.

  5. Endothelial cell high-enrichment from endovascular biopsy sample by laser capture microdissection and fluorescence activated cell sorting

    Science.gov (United States)

    Sun, Zhengda; Su, Hua; Long, Brian; Sinclair, Elizabeth; Hetts, Steven W.; Higashida, Randall T.; Dowd, Christopher F.; Halbach, Van V.; Cooke, Daniel L.

    2015-01-01

    Background and purpose Endovascular sampling and characterization from patients can provide very useful information about the pathogenesis of different vascular diseases, but it has been limited by the lack of an effective method of endothelial cell (EC) enrichment. We optimized the EC yield and enrichment from conventional guide wires by laser capture microdissection (LCM) and fluorescence activated cell sorting (FACS) technique, and addressed the feasibility of using these enriched ECs for downstream gene expression detection. Methods Iliac artery endovascular samples from 10 patients undergoing routine catheter angiography were collected using conventional 0.038 in. J-shape guide wires. Each of these samples was equally divided into two parts, which were respectively used for EC enrichment by immunocytochemistry-coupled LCM or multiple color FACS. After RNA extraction and reverse transcription, the amplified cDNA was used for quantitative polymerase chain reaction (qPCR). Results Fixed ECs, with positive CD31 or vWF fluorescent signal and endothelial like nucleus, were successfully separated by LCM and live single ECs were sorted on FACS by a seven color staining panel. EC yields by LCM and FACS were 51 ± 22 and 149 ± 56 respectively (P < 0.001). The minimum number of fixed ECs from ICC-coupled LCM for acceptable qPCR results of endothelial marker genes was 30, while acceptable qPCR results as enriched by FACS were attainable from a single live EC. Conclusion Both LCM and FACS can be used to enrich ECs from conventional guide wires and the enriched ECs can be used for downstream gene expression detection. FACS generated a higher EC yield and the sorted live ECs may be used for single cell gene expression detection. PMID:25450638

  6. The ROCK inhibitor Y-27632 improves recovery of human embryonic stem cells after fluorescence-activated cell sorting with multiple cell surface markers.

    Directory of Open Access Journals (Sweden)

    Nil Emre

    Full Text Available BACKGROUND: Due to the inherent sensitivity of human embryonic stem cells (hESCs to manipulations, the recovery and survival of hESCs after fluorescence-activated cell sorting (FACS can be low. Additionally, a well characterized and robust methodology for performing FACS on hESCs using multiple-cell surface markers has not been described. The p160-Rho-associated coiled kinase (ROCK inhibitor, Y-27632, previously has been identified as enhancing survival of hESCs upon single-cell dissociation, as well as enhancing recovery from cryopreservation. Here we examined the application of Y-27632 to hESCs after FACS to improve survival in both feeder-dependent and feeder-independent growth conditions. METHODOLOGY/PRINCIPAL FINDINGS: HESCs were sorted using markers for SSEA-3, TRA-1-81, and SSEA-1. Cells were plated after sorting for 24 hours in either the presence or the absence of Y-27632. In both feeder-dependent and feeder-independent conditions, cell survival was greater when Y-27632 was applied to the hESCs after sort. Specifically, treatment of cells with Y-27632 improved post-sort recovery up to four fold. To determine the long-term effects of sorting with and without the application of Y-27632, hESCs were further analyzed. Specifically, hESCs sorted with and without the addition of Y-27632 retained normal morphology, expressed hESC-specific markers as measured by immunocytochemistry and flow cytometry, and maintained a stable karyotype. In addition, the hESCs could differentiate into three germ layers in vitro and in vivo in both feeder-dependent and feeder-independent growth conditions. CONCLUSIONS/SIGNIFICANCE: The application of Y-27632 to hESCs after cell sorting improves cell recovery with no observed effect on pluripotency, and enables the consistent recovery of hESCs by FACS using multiple surface markers. This improved methodology for cell sorting of hESCs will aid many applications such as removal of hESCs from secondary cell types

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

    Directory of Open Access Journals (Sweden)

    Saurabh Singh

    2005-01-01

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

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

    Science.gov (United States)

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

    2011-04-01

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

  9. Flow cytometry and cell sorting.

    Science.gov (United States)

    Ibrahim, Sherrif F; van den Engh, Ger

    2007-01-01

    Flow cytometry and cell sorting are well-established technologies in clinical diagnostics and biomedical research. Heterogeneous mixtures of cells are placed in suspension and passed single file across one or more laser interrogation points. Light signals emitted from the particles are collected and correlated to entities such as cell morphology, surface and intracellular protein expression, gene expression, and cellular physiology. Based on user-defined parameters, individual cells can then be diverted from the fluid stream and collected into viable, homogeneous fractions at exceptionally high speeds and a purity that approaches 100%. As such, the cell sorter becomes the launching point for numerous downstream studies. Flow cytometry is a cornerstone in clinical diagnostics, and cheaper, more versatile machines are finding their way into widespread and varied uses. In addition, advances in computing and optics have led to a new generation of flow cytometers capable of processing cells at orders of magnitudes faster than their predecessors, and with staggering degrees of complexity, making the cytometer a powerful discovery tool in biotechnology. This chapter will begin with a discussion of basic principles of flow cytometry and cell sorting, including a technical description of factors that contribute to the performance of these instruments. The remaining sections will then be divided into clinical- and research-based applications of flow cytometry and cell sorting, highlighting salient studies that illustrate the versatility of this indispensable technology.

  10. Fluorescence-activated cell analysis and sorting of viable mammalian cells based on beta-D-galactosidase activity after transduction of Escherichia coli lacZ.

    Science.gov (United States)

    Nolan, G P; Fiering, S; Nicolas, J F; Herzenberg, L A

    1988-01-01

    We demonstrate that individual cells infected with and expressing a recombinant retrovirus carrying the Escherichia coli beta-galactosidase gene (lacZ) can be viably stained, analyzed, sorted, and cloned by fluorescence-activated cell sorting based on the levels of lacZ expressed. To accomplish this we have devised a method to enzymatically generate and maintain fluorescence in live mammalian cells. Accumulation of fluorescent products in cells is linear with time, with a direct correlation of fluorescence to enzymatic activity. This technology for beta-galactosidase detection is more sensitive than other available cytochemical or biochemical methods. We have used this procedure to show that the expression of psi-2-MMuLVSVnlsLacZ in the T-cell lymphoma BW5147 and the B-cell hybridoma SP2/0 is not completely stable and that subclones selected by the fluorescence-activated cell sorter for low lacZ activity demonstrate distinctly lower average expression of LacZ. These findings indicate the utility of beta-galactosidase as a reporter molecule at the single-cell level for studies of gene regulation, including studies of promoter efficacy, enhancer activity, trans-acting factors, and other regulatory elements. Images PMID:3128790

  11. A fluorescence-activated cell sorting-based strategy for rapid isolation of high-lipid Chlamydomonas mutants.

    Science.gov (United States)

    Terashima, Mia; Freeman, Elizabeth S; Jinkerson, Robert E; Jonikas, Martin C

    2015-01-01

    There is significant interest in farming algae for the direct production of biofuels and valuable lipids. Chlamydomonas reinhardtii is the leading model system for studying lipid metabolism in green algae, but current methods for isolating mutants of this organism with a perturbed lipid content are slow and tedious. Here, we present the Chlamydomonas high-lipid sorting (CHiLiS) strategy, which enables enrichment of high-lipid mutants by fluorescence-activated cell sorting (FACS) of pooled mutants stained with the lipid-sensitive dye Nile Red. This method only takes 5 weeks from mutagenesis to mutant isolation. We developed a staining protocol that allows quantification of lipid content while preserving cell viability. We improved separation of high-lipid mutants from the wild type by using each cell's chlorophyll fluorescence as an internal control. We initially demonstrated 20-fold enrichment of the known high-lipid mutant sta1 from a mixture of sta1 and wild-type cells. We then applied CHiLiS to sort thousands of high-lipid cells from a pool of about 60,000 mutants. Flow cytometry analysis of 24 individual mutants isolated by this approach revealed that about 50% showed a reproducible high-lipid phenotype. We further characterized nine of the mutants with the highest lipid content by flame ionization detection and mass spectrometry lipidomics. All mutants analyzed had a higher triacylglycerol content and perturbed whole-cell fatty acid composition. One arbitrarily chosen mutant was evaluated by microscopy, revealing larger lipid droplets than the wild type. The unprecedented throughput of CHiLiS opens the door to a systems-level understanding of green algal lipid biology by enabling genome-saturating isolation of mutants in key genes.

  12. Comparative analysis of mitosis-specific antibodies for bulk purification of mitotic populations by fluorescence-activated cell sorting.

    Science.gov (United States)

    Campbell, Amy E; Hsiung, Chris C-S; Blobel, Gerd A

    2014-01-01

    Mitosis entails complex chromatin changes that have garnered increasing interest from biologists who study genome structure and regulation-fields that are being advanced by high-throughput sequencing (Seq) technologies. The application of these technologies to study the mitotic genome requires large numbers of highly pure mitotic cells, with minimal contamination from interphase cells, to ensure accurate measurement of phenomena specific to mitosis. Here, we optimized a fluorescence-activated cell sorting (FACS)-based method for isolating formaldehyde-fixed mitotic cells--at virtually 100% mitotic purity and in quantities sufficient for high-throughput genomic studies. We compared several commercially available antibodies that react with mitosis-specific epitopes over a range of concentrations and cell numbers, finding antibody MPM2 to be the most robust and cost-effective.

  13. Polarized sorting and trafficking in epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Xinwang Cao; Michal A Surma; Kai Simons

    2012-01-01

    The polarized distribution of proteins and lipids at the surface membrane of epithelial cells results in the formation of an apical and a basolateral domain,which are separated by tight junctions.The generation and maintenance of epithelial polarity require elaborate mechanisms that guarantee correct sorting and vectorial delivery of cargo molecules.This dynamic process involves the interaction of sorting signals with sorting machineries and the formation of transport carriers.Here we review the recent advances in the field of polarized sorting in epithelial cells.We especially highlight the role of lipid rafts in apical sorting.

  14. Magnetic-activated cell sorting (MACS) can be used as a large-scale method for establishing zebrafish neuronal cell cultures

    OpenAIRE

    Georg Welzel; Daniel Seitz; Stefan Schuster

    2015-01-01

    Neuronal cell cultures offer a crucial tool to mechanistically analyse regeneration in the nervous system. Despite the increasing importance of zebrafish (Danio rerio) as an in vivo model in neurobiological and biomedical research, in vitro approaches to the nervous system are lagging far behind and no method is currently available for establishing enriched neuronal cell cultures. Here we show that magnetic-activated cell sorting (MACS) can be used for the large-scale generation of neuronal-r...

  15. Specific Single-Cell Isolation of Escherichia coli O157 from Environmental Water Samples by Using Flow Cytometry and Fluorescence-Activated Cell Sorting.

    Science.gov (United States)

    Ozawa, Shuji; Okabe, Satoshi; Ishii, Satoshi

    2016-08-01

    Contamination of food and water with pathogenic bacteria is of concern. Although culture-independent detection and quantification of pathogens is useful, isolation of pathogenic bacteria is still important when identifying the sources of pathogens. Here, we report the use of flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) to specifically detect and isolate individual Escherichia coli O157:H7 cells from water samples. When present at >10 cells/mL water, target pathogen was specifically detected and isolated. The FACS-sorted E. coli O157:H7 population reflected the original population diversity, in contrast to the populations obtained by immunomagnetic separation. Relative abundance of multiple pathogenic strains is important when performing source-tracking studies; therefore, single-cell isolation with FCM-FACS can be a useful tool to obtain pathogenic bacteria for source tracking purpose.

  16. High salt buffer improves integrity of RNA after fluorescence-activated cell sorting of intracellular labeled cells.

    Science.gov (United States)

    Nilsson, Helén; Krawczyk, Krzysztof M; Johansson, Martin E

    2014-12-20

    Over the past years, massive progress has been made in the ability to collect large-scale gene expression data from a limited sample size. Combined with improvements in multiplex flow cytometry-based techniques, this has made it possible to isolate and characterize specific cellular subtypes within heterogeneous populations, with a great impact on our understanding of different biological processes. However, sorting based on intracellular markers requires fixation and permeabilization of samples, and very often the integrity of RNA molecules is compromised during this process. Many attempts have been made to improve the quality of nucleic acids from such samples, but RNA degradation still remains a limiting factor for downstream analyses. Here we present a method to isolate high quality RNA from cells that have been fixed, permeabilized, intracellularly labeled and sorted. By performing all incubation steps in the presence of a high salt buffer, RNA degradation was avoided and samples with remarkable integrity were obtained. This procedure offers a straightforward and very affordable technique to retrieve high quality RNA from isolated cell populations, which increases the possibilities to characterize gene expression profiles of subpopulations from mixed samples, a technique with implications in a broad range of research fields.

  17. Recent advances in flow cytometric cell sorting.

    Science.gov (United States)

    Osborne, Geoffrey W

    2011-01-01

    The classification and separation of one cell type or particle from others is a fundamental task in many areas of science. Numerous techniques are available to perform this task; however, electrostatic cell sorting has gained eminence over others because, when combined with the analysis capabilities of flow cytometry it provides flexible separations based on multiple parameters. Unlike competing technologies, such as gradient or magnetic separations that offer much larger total throughput, flow cytometric cell sorting permits selections based on various levels of fluorescent reporters, rather the complete presence or absence of the reporter. As such, this technology has found application in a huge range of fields. This chapter aims to describe the utility of single-cell sorting with particular emphasis given to index sorting. This is followed by two recently developed novel techniques of sorting cells or particles. The first of these is positional sorting which is useful in cell-based studies where sorting can proceed and produce meaningful results without being inherently dependant on prior knowledge of where gates should be set. Secondly, reflective plate sorting is introduced which positionally links multiwell sample and collection plates in a convenient assay format so that cells in the collection plate "reflect" those in the sample plate.

  18. Comparison The Effects of Two Monocyte Isolation Methods,Plastic Adherence and Magnetic Activated Cell Sorting Methods,on Phagocytic Activity of Generated Dendritic Cells

    Directory of Open Access Journals (Sweden)

    Behnaz Asadi

    2013-01-01

    Full Text Available Objective: It is believed that monocyte isolation methods and maturation factors affect the phenotypic and functional characteristics of resultant dendritic cells (DC. In the present study, we compared two monocyte isolation methods, including plastic adherence-dendritic cells (Adh-DC and magnetic activated cell sorting- dendritic cells (MACS-DC, and their effects on phagocytic activity of differentiated immature DCs (immDCs.Materials and Methods: In this experimental study, immDCs were generated from plastic adherence and MACS isolated monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF and interleukin 4 (IL-4 in five days. The phagocytic activity of immDCs was analyzed by fluorescein isothiocyanate (FITC-conjugated latex bead using flow cytometry. One way ANOVA test was used for statistical analysis of differences among experimental groups, including Adh-DC and MACS-DC groups.Results: We found that phagocytic activity of Adh-DC was higher than MACS-DC, whereas the mean fluorescence intensity (MFI of phagocytic cells was higher in MACS-DC (p<0.05.Conclusion: We concluded that it would be important to consider phagocytosis parameters of generated DCs before making any decision about monocyte isolation methods to have fully functional DCs.

  19. Magnetic-activated cell sorting of TCR-engineered T cells, using tCD34 as a gene marker, but not peptide-MHC multimers, results in significant numbers of functional CD4+ and CD8+ T cells.

    Science.gov (United States)

    Govers, Coen; Berrevoets, Cor; Treffers-Westerlaken, Elike; Broertjes, Marieke; Debets, Reno

    2012-06-01

    T cell-sorting technologies with peptide-MHC multimers or antibodies against gene markers enable enrichment of antigen-specific T cells and are expected to enhance the therapeutic efficacy of clinical T cell therapy. However, a direct comparison between sorting reagents for their ability to enrich T cells is lacking. Here, we compared the in vitro properties of primary human T cells gene-engineered with gp100(280-288)/HLA-A2-specific T cell receptor-αβ (TCRαβ) on magnetic-activated cell sorting (MACS) with various peptide-MHC multimers or an antibody against truncated CD34 (tCD34). With respect to peptide-MHC multimers, we observed that Streptamer(®), when compared with pentamers and tetramers, improved T cell yield as well as level and stability of enrichment, of TCR-engineered T cells (>65% of peptide-MHC-binding T cells, stable for at least 6 weeks). In agreement with these findings, Streptamer, the only detachable reagent, revealed significant T cell expansion in the first week after MACS. Sorting TCR and tCD34 gene-engineered T cells with CD34 monoclonal antibody (mAb) resulted in the most significant T cell yield and enrichment of T cells (>95% of tCD34 T cells, stable for at least 6 weeks). Notably, T cells sorted with CD34 mAb, when compared with Streptamer, bound about 2- to 3-fold less peptide-MHC but showed superior antigen-specific upregulated expression of CD107a and production of interferon (IFN)-γ. Multiparametric flow cytometry revealed that CD4(+) T cells, uniquely present in CD34 mAb-sorted T cells, contributed to enhanced IFN-γ production. Taken together, we postulate that CD34 mAb-based sorting of gene-marked T cells has benefits toward applications of T cell therapy, especially those that require CD4(+) T cells.

  20. Laser ablation cell sorting in scanning cytometry

    Science.gov (United States)

    Shen, Feimo; Price, Jeffrey H.

    2001-05-01

    Flow cytometry has been an important tool for automated cells sorting. However, the lack of good sensitivity prevents it from being used for rare events sorting; furthermore, fragile cells, anchorage-dependent cells, and clump forming cells cannot be sorted this way. A fully automated, high-speed scanning cytometer with autofocus and image segmentation is capable of accurately locating contaminant cells in a monolayer cell population. A laser ablation system was incorporated into the cytometer to negatively sort out the unwanted cells by applying a focused, ultra-short laser pulse (sub-micron diameter, pulse duration = 4 nsec, wavelength - 500 nm) to each targeted cell. Due to the high power density (approximately 1010 W/cm2) that was present at the focal point, disruptive mechanical forces were generated and were responsible for the kill. Fluorescently stained NIH-3T3 fibroblast cells were used as a model contaminant target ells in an unstained NIH-3T3 population to determine the identification-kill effectiveness. The contaminant cells were stained with the fluorochrome CellTracker Blue CMAC, whereas the background cells were left intact. Ablation pulses were applied in frame-by-frame increment batches to the cell culture on the microscope. The negative sorting effectiveness was analyzed by automatically re-scanning the post-ablation cell culture in phase contrast and propidium iodide stained epi fluorescent fields to verify cell death.

  1. Noninvasive prenatal diagnosis. Use of density gradient centrifugation, magnetically activated cell sorting and in situ hybridization

    DEFF Research Database (Denmark)

    Campagnoli, C; Multhaupt, H A; Ludomirski, A;

    1997-01-01

    cells recovered did not differ. Seven of seven male pregnancies were correctly identified. One case of trisomy 21 was detected. CONCLUSION: The in situ hybridization analysis of fetal nucleated erythrocytes isolated from maternal blood using single density gradient centrifugation, anti-CD71/anti...... of the isolated cells were subjected to in situ hybridization with specific DNA probes for the Y chromosome and chromosome 21 to confirm the fetal origin. RESULTS: After MiniMACS the enrichment factors for the CD71/GPA- and CD36/GPA-positive cells from maternal blood were similar, and the percentages of fetal...

  2. Identification of microbes from the surfaces of food-processing lines based on the flow cytometric evaluation of cellular metabolic activity combined with cell sorting.

    Science.gov (United States)

    Juzwa, W; Duber, A; Myszka, K; Białas, W; Czaczyk, K

    2016-09-01

    In this study the design of a flow cytometry-based procedure to facilitate the detection of adherent bacteria from food-processing surfaces was evaluated. The measurement of the cellular redox potential (CRP) of microbial cells was combined with cell sorting for the identification of microorganisms. The procedure enhanced live/dead cell discrimination owing to the measurement of the cell physiology. The microbial contamination of the surface of a stainless steel conveyor used to process button mushrooms was evaluated in three independent experiments. The flow cytometry procedure provided a step towards monitoring of contamination and enabled the assessment of microbial food safety hazards by the discrimination of active, mid-active and non-active bacterial sub-populations based on determination of their cellular vitality and subsequently single cell sorting to isolate microbial strains from discriminated sub-populations. There was a significant correlation (r = 0.97; p flow cytometry, despite there being differences in the absolute number of cells detected. The combined approach of flow cytometric CRP measurement and cell sorting allowed an in situ analysis of microbial cell vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells.

  3. Minimal residual disease surveillance in chronic lymphocytic leukemia by fluorescence-activated cell sorting.

    Science.gov (United States)

    Ringelstein-Harlev, Shimrit; Fineman, Riva

    2014-10-01

    Achievement of complete response (CR) to therapy in chronic lymphocytic leukemia (CLL) has become a feasible goal, directly correlating with prolonged survival. It has been established that the classic definition of CR actually encompasses a variety of disease loads, and more sensitive multiparameter flow cytometry and polymerase chain reaction methods can detect the disease burden with a much higher sensitivity. Detection of malignant cells with a sensitivity of 1 tumor cell in 10,000 cells (10(-4)), using the abovementioned sophisticated techniques, is the current cutoff for minimal residual disease (MRD). Tumor burdens lower than 10(-4) are defined as MRD-negative. Several studies in CLL have determined the achievement of MRD negativity as an independent favorable prognostic factor, leading to prolonged disease-free and overall survival, regardless of the treatment protocol or the presence of other pre-existing prognostic indicators. Minimal residual disease evaluation using flow cytometry is a sensitive and applicable approach which is expected to become an integral part of future prospective trials in CLL designed to assess the role of MRD surveillance in treatment tailoring.

  4. Minimal Residual Disease Surveillance in Chronic Lymphocytic Leukemia by Fluorescence-Activated Cell Sorting

    Directory of Open Access Journals (Sweden)

    Shimrit Ringelstein-Harlev

    2014-10-01

    Full Text Available Achievement of complete response (CR to therapy in chronic lymphocytic leukemia (CLL has become a feasible goal, directly correlating with prolonged survival. It has been established that the classic definition of CR actually encompasses a variety of disease loads, and more sensitive multiparameter flow cytometry and polymerase chain reaction methods can detect the disease burden with a much higher sensitivity. Detection of malignant cells with a sensitivity of 1 tumor cell in 10,000 cells (10–4, using the abovementioned sophisticated techniques, is the current cutoff for minimal residual disease (MRD. Tumor burdens lower than 10–4 are defined as MRD-negative. Several studies in CLL have determined the achievement of MRD negativity as an independent favorable prognostic factor, leading to prolonged disease-free and overall survival, regardless of the treatment protocol or the presence of other pre-existing prognostic indicators. Minimal residual disease evaluation using flow cytometry is a sensitive and applicable approach which is expected to become an integral part of future prospective trials in CLL designed to assess the role of MRD surveillance in treatment tailoring.

  5. Applications of cell sorting in biotechnology

    Directory of Open Access Journals (Sweden)

    Mattanovich Diethard

    2006-03-01

    Full Text Available Abstract Due to its unique capability to analyze a large number of single cells for several parameters simultaneously, flow cytometry has changed our understanding of the behavior of cells in culture and of the population dynamics even of clonal populations. The potential of this method for biotechnological research, which is based on populations of living cells, was soon appreciated. Sorting applications, however, are still less frequent than one would expect with regard to their potential. This review highlights important contributions where flow cytometric cell sorting was used for physiological research, protein engineering, cell engineering, specifically emphasizing selection of overproducing cell lines. Finally conclusions are drawn concerning the impact of cell sorting on inverse metabolic engineering and systems biology.

  6. Isolation of myeloid-derived suppressor cells subsets from spleens of orthotopic liver cancer-bearing mice by fluorescent-activated and magnetic-activated cell sorting: similarities and differences.

    Science.gov (United States)

    Xu, Yaping; Zhao, Wenxiu; Wu, Duan; Xu, Jianfeng; Lin, Suqiong; Tang, Kai; Yin, Zhenyu; Wang, Xiaomin

    2014-01-01

    Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that commonly expand during tumor development and that play a critical role in suppression of immune responses. MDSCs can be classified into two groups: Mo-MDSCs and G-MDSCs. These cells differ in their morphology, phenotype, differentiation ability, and immunosuppressive activity, and inhibit immune responses via different mechanisms. Therefore, identifying an effective method for isolating viable Mo-MDSCs and G-MDSCs is important. Here, we demonstrated the differences and similarities between fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) in sorting G-MDSCs and Mo-MDSCs. Both MACS and FACS could obtain G-MDSCs and Mo-MDSCs with high viability and purity. A high yield and purity of G-MDSCs could be obtained both by using FACS and MACS, because G-MDSCs are highly expressed in the spleen of tumor-bearing mice. However, Mo-MDSCs, which comprise a small population among leukocytes, when sorted by MACS, could be obtained at much greater cell number, although with a slightly lower purity, than when sorted by FACS. In conclusion, we recommended using both FACS and MACS for isolating G-MDSCs, and using MACS for isolation of Mo-MDSCs.

  7. Optical cell sorting with multiple imaging modalities

    DEFF Research Database (Denmark)

    Banas, Andrew; Carrissemoux, Caro; Palima, Darwin

    2017-01-01

    techniques. Scattering forces from beams actuated via efficient phase-only efficient modulation has been adopted. This has lowered the required power for sorting cells to a tenth of our previous approach, and also makes the cell sorter safer for use in clinical settings. With the versatility of dynamically...... programmable phase spatial light modulators, a plurality of light shaping techniques, including hybrid approaches, can be utilized in cell sorting....... healthy cells. With the richness of visual information, a lot of microscopy techniques have been developed and have been crucial in biological studies. To utilize their complementary advantages we adopt both fluorescence and brightfield imaging in our optical cell sorter. Brightfield imaging has...

  8. Preparation of myeloid derived suppressor cells (MDSC) from naive and pancreatic tumor-bearing mice using flow cytometry and automated magnetic activated cell sorting (AutoMACS).

    Science.gov (United States)

    Nelson, Nadine; Szekeres, Karoly; Cooper, Denise; Ghansah, Tomar

    2012-06-18

    MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological conditions including parasitic infection, inflammation, traumatic stress, graft-versus-host disease, diabetes and cancer. In mice, MDSC express Mac-1 (CD11b) and Gr-1 (Ly6G and Ly6C) surface antigens. It is important to note that MDSC are well studied in various tumor-bearing hosts where they are significantly expanded and suppress anti-tumor immune responses compared to naïve counterparts. However, depending on the pathological condition, there are different subpopulations of MDSC with distinct mechanisms and targets of suppression. Therefore, effective methods to isolate viable MDSC populations are important in elucidating their different molecular mechanisms of suppression in vitro and in vivo. Recently, the Ghansah group has reported the expansion of MDSC in a murine pancreatic cancer model. Our tumor-bearing MDSC display a loss of homeostasis and increased suppressive function compared to naïve MDSC. MDSC percentages are significantly less in lymphoid compartments of naïve vs. tumor-bearing mice. This is a major caveat, which often hinders accurate comparative analyses of these MDSC. Therefore, enriching Gr-1(+) leukocytes from naïve mice prior to Fluorescence Activated Cell Sorting (FACS) enhances purity, viability and significantly reduces sort time. However, enrichment of Gr-1(+) leukocytes from tumor-bearing mice is optional as these are in abundance for quick FACS sorting. Therefore, in this protocol, we describe a highly efficient method of immunophenotyping MDSC and enriching Gr-1(+) leukocytes from spleens of naïve mice for sorting MDSC in a timely manner. Immunocompetent C57BL/6 mice are inoculated with murine Panc02 cells subcutaneously whereas naïve mice receive 1XPBS. Approximately 30 days post inoculation; spleens are harvested and processed into single-cell suspensions using a cell dissociation

  9. Sorting cells by their dynamical properties

    Science.gov (United States)

    Henry, Ewan; Holm, Stefan H.; Zhang, Zunmin; Beech, Jason P.; Tegenfeldt, Jonas O.; Fedosov, Dmitry A.; Gompper, Gerhard

    2016-10-01

    Recent advances in cell sorting aim at the development of novel methods that are sensitive to various mechanical properties of cells. Microfluidic technologies have a great potential for cell sorting; however, the design of many micro-devices is based on theories developed for rigid spherical particles with size as a separation parameter. Clearly, most bioparticles are non-spherical and deformable and therefore exhibit a much more intricate behavior in fluid flow than rigid spheres. Here, we demonstrate the use of cells’ mechanical and dynamical properties as biomarkers for separation by employing a combination of mesoscale hydrodynamic simulations and microfluidic experiments. The dynamic behavior of red blood cells (RBCs) within deterministic lateral displacement (DLD) devices is investigated for different device geometries and viscosity contrasts between the intra-cellular fluid and suspending medium. We find that the viscosity contrast and associated cell dynamics clearly determine the RBC trajectory through a DLD device. Simulation results compare well to experiments and provide new insights into the physical mechanisms which govern the sorting of non-spherical and deformable cells in DLD devices. Finally, we discuss the implications of cell dynamics for sorting schemes based on properties other than cell size, such as mechanics and morphology.

  10. Isolation, purification, culture and characterisation of myoepithelial cells from normal and neoplastic canine mammary glands using a magnetic-activated cell sorting separation system.

    Science.gov (United States)

    Sánchez-Céspedes, R; Maniscalco, L; Iussich, S; Martignani, E; Guil-Luna, S; De Maria, R; Martín de Las Mulas, J; Millán, Y

    2013-08-01

    Mammary gland tumours, the most common malignant neoplasm in bitches, often display myoepithelial (ME) cell proliferation. The aim of this study was to isolate, purify, culture and characterise ME cells from normal and neoplastic canine mammary glands. Monodispersed cells from three normal canine mammary glands and five canine mammary tumours were incubated with an anti-Thy1 antibody and isolated by magnetic-activated cell sorting (MACS). Cells isolated from two normal glands (cell lines CmME-N1 and CmME-N2) and four tumours (cell lines CmME-K1 from a complex carcinoma, CmME-K2 from a simple tubulopapillary carcinoma, and CmME-K3 and CmME-K4 from two carcinomas within benign tumours) were cultured in supplemented DMEM/F12 media for 40days. Cell purity was >90%. Tumour-derived ME cell lines exhibited heterogeneous morphology, growth patterns and immunocytochemical expression of cytokeratins, whereas cell lines from normal glands retained their morphology and levels of cytokeratin expression during culture. Cell lines from normal glands and carcinomas within benign tumours grew more slowly than those from simple and complex carcinomas. This methodology has the potential to be used for in vitro analysis of the role of ME cells in the growth and progression of canine mammary tumours.

  11. Integration through a Card-Sort Activity

    Science.gov (United States)

    Green, Kris; Ricca, Bernard P.

    2015-01-01

    Learning to compute integrals via the various techniques of integration (e.g., integration by parts, partial fractions, etc.) is difficult for many students. Here, we look at how students in a college level Calculus II course develop the ability to categorize integrals and the difficulties they encounter using a card sort-resort activity. Analysis…

  12. Integration through a Card-Sort Activity

    Science.gov (United States)

    Green, Kris; Ricca, Bernard P.

    2015-01-01

    Learning to compute integrals via the various techniques of integration (e.g., integration by parts, partial fractions, etc.) is difficult for many students. Here, we look at how students in a college level Calculus II course develop the ability to categorize integrals and the difficulties they encounter using a card sort-resort activity. Analysis…

  13. Machine-vision based optofluidic cell sorting

    DEFF Research Database (Denmark)

    Glückstad, Jesper; Bañas, Andrew

    In contemporary life science there is an increasing emphasis on sorting rare disease-indicating cells within small dilute quantities such as in the confines of optofluidic lab-on-chip devices. Our approach to this is based on the use of optical forces to isolate red blood cells detected by advanc...... the available light and creating 2D or 3D beam distributions aimed at the positions of the detected cells. Furthermore, the beam shaping freedom provided by GPC can allow optimizations in the beam’s propagation and its interaction with the laser catapulted and sorted cells....... machine vision1. This approach is gentler, less invasive and more economical compared to conventional FACS-systems. As cells are less responsive to plastic or glass objects commonly used in the optical manipulation literature2, and since laser safety would be an issue in clinical use, we develop efficient...

  14. Sex-sorting sperm using flow cytometry/cell sorting.

    Science.gov (United States)

    Garner, Duane L; Evans, K Michael; Seidel, George E

    2013-01-01

    The sex of mammalian offspring can be predetermined by flow sorting relatively pure living populations of X- and Y-chromosome-bearing sperm. This method is based on precise staining of the DNA of sperm with the nucleic acid-specific fluorophore, Hoechst 33342, to differentiate between the subpopulations of X- and Y-sperm. The fluorescently stained sperm are then sex-sorted using a specialized high speed sorter, MoFlo(®) SX XDP, and collected into biologically supportive media prior to reconcentration and cryopreservation in numbers adequate for use with artificial insemination for some species or for in vitro fertilization. Sperm sorting can provide subpopulations of X- or Y-bearing bovine sperm at rates in the 8,000 sperm/s range while maintaining; a purity of 90% such that it has been applied to cattle on a commercial basis. The sex of offspring has been predetermined in a wide variety of mammalian species including cattle, swine, horses, sheep, goats, dogs, cats, deer, elk, dolphins, water buffalo as well as in humans using flow cytometric sorting of X- and Y-sperm.

  15. Comparative analysis of circulating endothelial progenitor cells in age-related macular degeneration patients using automated rare cell analysis (ARCA and fluorescence activated cell sorting (FACS.

    Directory of Open Access Journals (Sweden)

    Emil Anthony T Say

    Full Text Available BACKGROUND: Patients with age-related macular degeneration (ARMD begin with non-neovascular (NNV phenotypes usually associated with good vision. Approximately 20% of NNV-ARMD patients will convert to vision debilitating neovascular (NV ARMD, but precise timing of this event is unknown. Developing a clinical test predicting impending conversion to NV-ARMD is necessary to prevent vision loss. Endothelial progenitor cells (EPCs, defined as CD34(+VEGR2(+ using traditional fluorescence activated cell sorting (FACS, are rare cell populations known to be elevated in patients with NV-ARMD compared to NNV-ARMD. FACS has high inter-observer variability and subjectivity when measuring rare cell populations precluding development into a diagnostic test. We hypothesized that automated rare cell analysis (ARCA, a validated and FDA-approved technology for reproducible rare cell identification, can enumerate EPCs in ARMD patients more reliably. This pilot study serves as the first step in developing methods for reproducibly predicting ARMD phenotype conversion. METHODS: We obtained peripheral venous blood samples in 23 subjects with NNV-ARMD or treatment naïve NV-ARMD. Strict criteria were used to exclude subjects with known angiogenic diseases to minimize confounding results. Blood samples were analyzed in masked fashion in two separate laboratories. EPCs were independently enumerated using ARCA and FACS within 24 hours of blood sample collection, and p<0.2 was considered indicative of a trend for this proof of concept study, while statistical significance was established at 0.05. RESULTS: We measured levels of CD34(+VEGFR2(+ EPCs suggestive of a trend with higher values in patients with NV compared to NNV-ARMD (p = 0.17 using ARCA. Interestingly, CD34(+VEGR2(+ EPC analysis using FACS did not produce similar results (p = 0.94. CONCLUSIONS: CD34(+VEGR2(+ may have predictive value for EPC enumeration in future ARCA studies. EPC measurements in a small sample

  16. Evaluating Effects of Cell Sorting on Cellular Integrity

    OpenAIRE

    2014-01-01

    During the past year the Flow Cytometry Research Group has continued on its goal to establish best practice guidelines for cell sorting conditions that minimize cell stress, perturbation, or injury to the sorted cells. Towards this goal the group has followed up on an observation from our initial study that showed poor cell recovery when a clonal population of cells (Jurkat) was sorted aggressively under intentionally adverse sorting conditions (excessive pressure as well as undersized sortin...

  17. How Schwann Cells Sort Axons: New Concepts.

    Science.gov (United States)

    Feltri, M Laura; Poitelon, Yannick; Previtali, Stefano Carlo

    2016-06-01

    Peripheral nerves contain large myelinated and small unmyelinated (Remak) fibers that perform different functions. The choice to myelinate or not is dictated to Schwann cells by the axon itself, based on the amount of neuregulin I-type III exposed on its membrane. Peripheral axons are more important in determining the final myelination fate than central axons, and the implications for this difference in Schwann cells and oligodendrocytes are discussed. Interestingly, this choice is reversible during pathology, accounting for the remarkable plasticity of Schwann cells, and contributing to the regenerative potential of the peripheral nervous system. Radial sorting is the process by which Schwann cells choose larger axons to myelinate during development. This crucial morphogenetic step is a prerequisite for myelination and for differentiation of Remak fibers, and is arrested in human diseases due to mutations in genes coding for extracellular matrix and linkage molecules. In this review we will summarize progresses made in the last years by a flurry of reverse genetic experiments in mice and fish. This work revealed novel molecules that control radial sorting, and contributed unexpected ideas to our understanding of the cellular and molecular mechanisms that control radial sorting of axons.

  18. Microtechnology for cell manipulation and sorting

    CERN Document Server

    Tseng, Peter; Carlo, Dino

    2017-01-01

    This book delves into the recent developments in the microscale and microfluidic technologies that allow manipulation at the single and cell aggregate level. Expert authors review the dominant mechanisms that manipulate and sort biological structures, making this a state-of-the-art overview of conventional cell sorting techniques, the principles of microfluidics, and of microfluidic devices. All chapters highlight the benefits and drawbacks of each technique they discuss, which include magnetic, electrical, optical, acoustic, gravity/sedimentation, inertial, deformability, and aqueous two-phase systems as the dominant mechanisms utilized by microfluidic devices to handle biological samples. Each chapter explains the physics of the mechanism at work, and reviews common geometries and devices to help readers decide the type of style of device required for various applications. This book is appropriate for graduate-level biomedical engineering and analytical chemistry students, as well as engineers and scientist...

  19. An improved protocol for mRNA quantification after fluorescence-activated cell sorting with an increased signal to noise ratio in flow cytometry.

    Science.gov (United States)

    Date, Arisa; Maeda, Tomoko; Watanabe, Mikio; Hidaka, Yoh; Iwatani, Yoshinori; Takano, Toru

    2014-07-01

    We established a method to analyze cells collected by fluorescence-activated cell sorting (FACS) named mRNA quantification after FACS (FACS-mQ), in which cells are labeled with a fluorescent dye in a manner that minimizes RNA degradation, and then cells sorted by FACS are examined by analyzing their gene expression profile. In this study, we established a modified protocol to analyze molecules with a low expression level, such as N-cadherin and thyroid transcription factor, by improving the signal to noise ratio in flow cytometry. Use of a fluorophore-conjugated second antibody and the appropriate choice of a fluorescence dye showed a marked increase in the signal to noise ratio. Use of the Can Get Signal Immunostain in diluting antibodies shortened the reaction time. In real-time reverse transcription-PCR, a significant decrease in the copy number of intracellular mRNAs was not observed after in-tube immunostaining. These results indicated that the present protocol is useful for separating and analyzing cells by FACS-mQ, targeting a molecule with a low expression level.

  20. Tracking heavy water (D2O) incorporation for identifying and sorting active microbial cells

    DEFF Research Database (Denmark)

    Berry, David; Mader, Esther; Lee, Tae Kwon;

    2015-01-01

    peaks in single-cell Raman spectra, and the obtained labeling pattern was confirmed by nanoscaleresolution secondary ion MS. In fast-growing Escherichia coli cells, label detection was already possible after 20 min. For functional analyses of microbial communities, the detection of D incorporation from...... D2O in individual microbial cells via Raman microspectroscopy can be directly combined with FISH for the identification of active microbes. Applying this approach to mouse cecal microbiota revealed that the host-compound foragers Akkermansia muciniphila and Bacteroides acidifaciens exhibited...

  1. Magnetic-Activated Cell Sorting of TCR-engineered T cells using tCD34 as a gene marker, but not peptide-MHC multimers, results in significant numbers of functional CD4 and CD8 T cells

    NARCIS (Netherlands)

    Govers, C.; Berrevoets, C.; Treffers-Westerlaken, E.; Broertjes, M.; Debets, R.

    2012-01-01

    T cell sorting technologies with peptide-MHC multimers or antibodies against gene markers enable enrichment of antigen-specific T cells and are expected to enhance therapeutic efficacy of clinical T cell therapy. However, a direct comparison between sorting reagents for their ability to enrich T cel

  2. Magnetic-activated cell sorting of TCR-engineered T cells, using tCD34 as a gene marker, but not peptide-MHC multimers, results in significant numbers of functional CD4+ and CD8+ T cells

    NARCIS (Netherlands)

    C.C.F.M. Govers (Coen); C.A. Berrevoets (Cor); E. Treffers-Westerlaken (Elike); M. Broertjes (Marieke); J.E.M.A. Debets (Reno)

    2012-01-01

    textabstractT cell-sorting technologies with peptide-MHC multimers or antibodies against gene markers enable enrichment of antigen-specific T cells and are expected to enhance the therapeutic efficacy of clinical T cell therapy. However, a direct comparison between sorting reagents for their ability

  3. Microfluidic-chip platform for cell sorting

    Science.gov (United States)

    Malik, Sarul; Balyan, Prerna; Akhtar, J.; Agarwal, Ajay

    2016-04-01

    Cell sorting and separation are considered to be very crucial preparatory steps for numerous clinical diagnostics and therapeutics applications in cell biology research arena. Label free cell separation techniques acceptance rate has been increased to multifold by various research groups. Size based cell separation method focuses on the intrinsic properties of the cell which not only avoids clogging issues associated with mechanical and centrifugation filtration methods but also reduces the overall cost for the process. Consequentially flow based cell separation method for continuous flow has attracted the attention of millions. Due to the realization of structures close to particle size in micro dimensions, the microfluidic devices offer precise and rapid particle manipulation which ultimately leads to an extraordinary cell separation results. The proposed microfluidic device is fabricated to separate polystyrene beads of size 1 µm, 5 µm, 10 µm and 20 µm. The actual dimensions of blood corpuscles were kept in mind while deciding the particle size of polystyrene beads which are used as a model particles for study.

  4. Viable cell sorting of dinoflagellates by multiparametric flow cytometry.

    Science.gov (United States)

    Sinigalliano, Christopher D; Winshell, Jamie; Guerrero, Maria A; Scorzetti, Gloria; Fell, Jack W; Eaton, Richard W; Brand, Larry; Rein, Kathleen S

    2009-07-01

    Electronic cell sorting for isolation and culture of dinoflagellates and other marine eukaryotic phytoplankton was compared to the traditional method of manually picking cells using a micropipette. Trauma to electronically sorted cells was not a limiting factor, as fragile dinoflagellates, such as Karenia brevis (Dinophyceae), survived electronic cell sorting to yield viable cells. The rate of successful isolation of large-scale (> 4 litres) cultures was higher for manual picking than for electronic cell sorting (2% vs 0.5%, respectively). However, manual picking of cells is more labor intensive and time consuming. Most manually isolated cells required repicking, as the cultures were determined not to be unialgal after a single round of isolation; whereas, no cultures obtained in this study from electronic single-cell sorting required resorting. A broad flow cytometric gating logic was employed to enhance species diversity. The percentages of unique genotypes produced by manual picking or electronic cell sorting were similar (57% vs 54%, respectively), and each approach produced a variety of dinoflagellate or raphidophyte genera. Alternatively, a highly restrictive gating logic was successfully used to target K. brevis from a natural bloom sample. Direct electronic single-cell sorting was more successful than utilizing a pre-enrichment sort followed by electronic single-cell sorting. The appropriate recovery medium may enhance the rate of successful isolations. Seventy percent of isolated cells were recovered in a new medium (RE) reported here, which was optimized for axenic dinoflagellate cultures. The greatest limiting factor to the throughput of electronic cell sorting is the need for manual postsort culture maintenance and assessment of the large number of isolated cells. However, when combined with newly developed automated methods for growth screening, electronic single-cell sorting has the potential to accelerate the discovery of new algal strains.

  5. Cell sorting using efficient light shaping approaches

    Science.gov (United States)

    Bañas, Andrew; Palima, Darwin; Villangca, Mark; Glückstad, Jesper

    2016-03-01

    Early detection of diseases can save lives. Hence, there is emphasis in sorting rare disease-indicating cells within small dilute quantities such as in the confines of lab-on-a-chip devices. In our work, we use optical forces to isolate red blood cells detected by machine vision. This approach is gentler, less invasive and more economical compared to conventional FACS systems. As cells are less responsive to plastic or glass beads commonly used in the optical manipulation literature, and since laser safety would be an issue in clinical use, we develop efficient approaches in utilizing lasers and light modulation devices. The Generalized Phase Contrast (GPC) method that can be used for efficiently illuminating spatial light modulators or creating well-defined contiguous optical traps is supplemented by diffractive techniques capable of integrating the available light and creating 2D or 3D beam distributions aimed at the positions of the detected cells. Furthermore, the beam shaping freedom provided by GPC can allow optimizations in the beam's propagation and its interaction with the catapulted cells.

  6. Prefrontal cell activities related to monkeys' success and failure in adapting to rule changes in a Wisconsin Card Sorting Test analog.

    Science.gov (United States)

    Mansouri, Farshad A; Matsumoto, Kenji; Tanaka, Keiji

    2006-03-08

    The cognitive flexibility to select appropriate rules in a changing environment is essential for survival and is assumed to depend on the integrity of prefrontal cortex (PFC). To explore the contribution of the dorsolateral PFC to flexible rule-based behavior, we recorded the activity of cells in this region of monkeys performing a Wisconsin Card Sorting Test (WCST) analog. The monkey had to match a sample to one of three test items by either color or shape. Liquid reward and a discrete visual signal (error signal) were given as feedback to correct and incorrect target selections, respectively. The relevant rule and its frequent changes were not cued, and the monkeys could find it only by interpreting the feedback. In one-third of cells, cellular activity was modulated by the relevant rule, both throughout the trial and between trials. The magnitude of the modulation correlated with the number of errors that the monkeys committed after each rule change in the course of reestablishing high performance. Activity of other cells differed between correct and error trials independently from the rule-related modulation. This difference appeared during actual responses and before the monkeys faced the problems. Many PFC cells responded to the error-signal presentation, and, in some of them, the magnitude of response depended on the relevant rule. These results suggest that the dorsolateral PFC contributes to WCST performance by maintaining the relevant rule across trials, assessing behavioral outcomes, and monitoring the processes that could lead to success and failure in individual trials.

  7. Standard practice for cell sorting in a BSL-3 facility.

    Science.gov (United States)

    Perfetto, Stephen P; Ambrozak, David R; Nguyen, Richard; Roederer, Mario; Koup, Richard A; Holmes, Kevin L

    2011-01-01

    Over the past decade, there has been a rapid growth in the number of BSL-3 and BSL-4 laboratories in the USA and an increase in demand for infectious cell sorting in BSL-3 laboratories. In 2007, the International Society for Advancement of Cytometry (ISAC) Biosafety Committee published standards for the sorting of unfixed cells and is an important resource for biosafety procedures when performing infectious cell sorting. Following a careful risk assessment, if it is determined that a cell sorter must be located within a BSL-3 laboratory, there are a variety of factors to be considered prior to the establishment of the laboratory. This chapter outlines procedures for infectious cell sorting in a BSL-3 environment to facilitate the establishment and safe operation of a BSL-3 cell sorting laboratory. Subjects covered include containment verification, remote operation, disinfection, personal protective equipment (PPE), and instrument-specific modifications for enhanced aerosol evacuation.

  8. Multiparametric flow cytometry for identification and fluorescence activated cell sorting of five distinct B-cell subpopulations in normal tonsil tissue.

    Science.gov (United States)

    Kjeldsen, Malene Krag; Perez-Andres, Martin; Schmitz, Alexander; Johansen, Preben; Boegsted, Martin; Nyegaard, Mette; Gaihede, Michael; Bukh, Anne; Johnsen, Hans E; Orfao, Alberto; Dybkaer, Karen

    2011-12-01

    The purpose of this study was to establish a procedure capable of isolating distinct B-cell subpopulations from human tonsils as a basis for subsequent molecular analyses. Overall, 5 distinct B-cell subpopulations were purified from fresh tonsils based on their fluorescence surface marker expression: naive B cells, centroblasts, centrocytes, memory B cells, and plasmablasts. The immunophenotypic identity of the subpopulations was verified by quantitative real-time reverse transcriptase-polymerase chain reaction using the proliferation marker MKI-67 and 6 B-cell-associated differentiation markers (BACH2, BCL6, PAX5, IRF4, PRDM1, and XBP1). Furthermore, within the centroblast compartment, large and small centroblasts could be distinguished and large centroblasts were shown to proliferate with a morphologic appearance of a "centroblast"-like cell but with lower gene expression of the germinal center markers BCL6 and BACH2 vs small centroblasts. This study has established a detailed and fast procedure for simultaneous sorting of up to 5 distinct maturation-associated B-cell subpopulations from human tonsils.

  9. Optical sorting and photo-transfection of mammalian cells

    CSIR Research Space (South Africa)

    Mthunzi, P

    2010-02-01

    Full Text Available Recently, laser light sources of different regimes have emerged as an essential tool in the biophotonics research area. Classic applications include, for example: manipulating single cells and their subcellular organelles, sorting cells...

  10. Safe sorting of GFP-transduced live cells for subsequent culture using a modified FACS vantage

    DEFF Research Database (Denmark)

    Sørensen, T U; Gram, G J; Nielsen, S D

    1999-01-01

    BACKGROUND: A stream-in-air cell sorter enables rapid sorting to a high purity, but it is not well suited for sorting of infectious material due to the risk of airborne spread to the surroundings. METHODS: A FACS Vantage cell sorter was modified for safe use with potentially HIV infected cells. S...... culture. CONCLUSIONS: Sorting of live infected cells can be performed safely and with no deleterious effects on vector expression using the modified FACS Vantage instrument.......BACKGROUND: A stream-in-air cell sorter enables rapid sorting to a high purity, but it is not well suited for sorting of infectious material due to the risk of airborne spread to the surroundings. METHODS: A FACS Vantage cell sorter was modified for safe use with potentially HIV infected cells...... culture. RESULTS: The bacteriophage sorting showed that the biologically active material was confined to the sorting chamber. A failure mode simulating a nozzle blockage resulted in detectable droplets inside the sorting chamber, but no droplets could be detected when an additional air suction from...

  11. Mutant HbpR transcription activator isolation for 2-chlorobiphenyl via green fluorescent protein-based flow cytometry and cell sorting.

    Science.gov (United States)

    Beggah, Siham; Vogne, Christelle; Zenaro, Elena; Van Der Meer, Jan Roelof

    2008-01-01

    Mutants were produced in the A-domain of HbpR, a protein belonging to the XylR family of σ(54)-dependent transcription activators, with the purpose of changing its effector recognition specificity from 2-hydroxybiphenyl (2-HBP, the cognate effector) to 2-chlorobiphenyl (2-CBP). Mutations were introduced in the hbpR gene part for the A-domain via error-prone polymerase chain reaction, and assembled on a gene circuitry plasmid in Escherichia coli, permitting HbpR-dependent induction of the enhanced green fluorescent protein (egfp). Cells with mutant HbpR proteins responsive to 2-CBP were enriched and separated in a flow cytometry-assisted cell-sorting procedure. Some 70 mutants were isolated and the A-domain mutations mapped. One of these had acquired true 2-CBP recognition but reacted hypersensitively to 2-HBP (20-fold more than the wild type), whereas others had reduced sensitivity to 2-HBP but a gain of 2-CBP recognition. Sequencing showed that most mutants carried double or triple mutations in the A-domain gene part, and were not located in previously recognized conserved residues within the XylR family members. Further selection from a new mutant pool prepared of the hypersensitive mutant did not result in increased 2-CBP or reduced 2-HBP recognition. Our data thus demonstrate that a one-step in vitro 'evolutionary' adaptation of the HbpR protein can result in both enhancement and reduction of the native effector recognition.

  12. Immunophenotypic comparison of heterogenous non-sorted versus sorted mononuclear cells from human umbilical cord blood: a novel cell enrichment approach.

    Science.gov (United States)

    Indumathi, S; Harikrishnan, R; Rajkumar, J S; Dhanasekaran, M

    2015-01-01

    Human umbilical cord blood (hUCB) has been the preferred source of stem cells for the treatment of haematological malignancies and genetic disorders. This is primarily due to its non-invasiveness, high accessibility with relative ease of isolation. Still failures do prevail due to its heterogeneity and lesser frequency of MSC identified in UCB. This study, thus, employs a cell enrichment technology to improve its therapeutic efficacy. This was achieved by immunophenotypic comparison of stem cells isolated from the heterogenous non-sorted mononuclear cells (MNCs), linage depleted (Lin+ and Lin-) fractions obtained from magnetic activated cell sorter (MACS) and sorted MNCs obtained by fluorescent activated cell sorter (FACS). The markers under consideration were CD29, CD44, CD34, CD45, CD133, CD90 and CD117. FACS sorted MNCs were rich in naive stem cell population, whereas non-sorted MNCs and lineage depleted fractions were found to be rich in progenitors. Thus, we suggest that a combination therapy of both sorted population might serve as an alternative valuable tool in treating haematologic/genetic disorders. However, further research on cell enrichment technology might give a clue for improved cell based therapy in regenerative medicine.

  13. Msh homeobox genes regulate cadherin-mediated cell adhesion and cell-cell sorting.

    Science.gov (United States)

    Lincecum, J M; Fannon, A; Song, K; Wang, Y; Sassoon, D A

    1998-07-01

    Msx-1 and Msx-2 are two closely related homeobox genes expressed in cephalic neural crest tooth buds, the optic cup endocardial cushions, and the developing limb [Hill and Davidson, 1991; Monaghan et al., 1991; Robert et al., 1991]. These sites correspond to regions of active cell segregation and proliferation under the influence of epithelial-mesenchymal cell interactions [Brown et al., 1993; Davidson et al., 1991], suggesting that Msx-1 and Msx-2 regulate cell-cell interactions. We have investigated the potential relationship between expression of the Msh homeobox genes (Msx-1 and Msx-2) and cadherin-mediated cell adhesion and cell sorting. We report that cell lines stably expressing Msx-1 or Msx-2 differentially sort on the basis of Msh gene expression. We demonstrate in vitro that initial cell aggregation involves calcium-dependent adhesion molecules (cadherins) and that Msh genes regulate cadherin-mediated adhesion. These results support the hypothesis that Msh genes play a role in the regulation of cell-cell adhesion and provide a link between the genetic phenomena of homeobox gene expression and cellular events involved in morphogenesis, including cell sorting and proliferation.

  14. Magnetic activated cell sorting: an effective method for reduction of sperm DNA fragmentation in varicocele men prior to assisted reproductive techniques.

    Science.gov (United States)

    Degheidy, T; Abdelfattah, H; Seif, A; Albuz, F K; Gazi, S; Abbas, S

    2015-10-01

    Semen parameters of varicocele men have been usually suspected to exhibit higher levels of abnormalities including DNA fragmentation, reactive oxygen species (ROS) and apoptotic markers. Negative correlation between increased level of DNA fragmentation and assisted reproductive techniques (ART) outcome has been studied by several authors. In the current study, we aim to evaluate the possible value of magnetic activated cell sorting (MACs) technology in reduction of DNA fragmentation in infertile varicocele patients prior to ART. Semen samples, collected from 36 varicocele patients, were prepared by density gradient centrifugation (DGC). Every sample was subsequently divided into two aliquots. One aliquot was kept untouched as pre-MACs control while the other aliquot was subjected to MACs technique, for depletion of apoptotic spermatozoa, and serves as post-MACs test. Sperm count, motility and DNA fragmentations were evaluated for both control and test samples. Post-MACs samples showed no deleterious reduction in total sperm motility (80.64 ± 6.97%) compared with control samples (80.97 ± 7.74%) while sperm DNA fragmentations were significantly reduced in post-MACs samples (9.61 ± 5.62%) compared with pre-MACs controls (12.43 ± 6.29%) (P DNA fragmentation in infertile varicocele patients prior to ART. © 2014 Blackwell Verlag GmbH.

  15. A cell sorting protocol for selecting high-producing sub-populations of Sf9 and High Five™ cells.

    Science.gov (United States)

    Vidigal, João; Dias, Mafalda M; Fernandes, Fabiana; Patrone, Marco; Bispo, Cláudia; Andrade, Cláudia; Gardner, Rui; Carrondo, Manuel J T; Alves, Paula M; Teixeira, Ana P

    2013-12-01

    Insect cell lines such as Sf9 and High Five™ have been widely used to produce recombinant proteins mostly by the lytic baculovirus vector system. We have recently established an expression platform in Sf9 cells using a fluorescence-based recombinase mediated cassette exchange (RMCE) strategy which has similar development timelines but avoids baculovirus infection. To expedite cell engineering efforts, a robust fluorescence-activated cell sorting (FACS) protocol optimized for insect cells was developed here. The standard sorting conditions used for mammalian cells proved to be unsuitable, resulting in post-sorting viabilities below 10% for both cell lines. We found that the extreme sensitivity to the shear stress displayed by Sf9 and High Five™ cells was the limiting factor, and using Pluronic F-68 in the cell suspension could increase post-sorting viabilities in a dose dependent manner. The newly developed protocol was then used to sort stable populations of both cell lines tagged with a DsRed-expressing cassette. Before sorting, the average fluorescence intensity of the Sf9 cell population was 3-fold higher than that of the High Five™ cell population. By enriching with the 10% strongest DsRed-fluorescent cells, the productivity of both cell populations could be successfully improved. The established sorting protocol potentiates the use of RMCE technology for recombinant protein production in insect cells.

  16. Efficient isolation of sperm with high DNA integrity and stable chromatin packaging by a combination of density-gradient centrifugation and magnetic-activated cell sorting.

    Science.gov (United States)

    Chi, Hee-Jun; Kwak, Su-Jin; Kim, Seok-Gi; Kim, Youn-Young; Park, Ji-Young; Yoo, Chang-Seok; Park, Il-Hae; Sun, Hong-Gil; Kim, Jae-Won; Lee, Kyeong-Ho

    2016-12-01

    This study was carried out to investigate the correlations of the sperm DNA fragmentation index (DFI) with semen parameters and apoptosis, and to investigate the effects of density-gradient centrifugation (DGC) and magnetic-activated cell sorting (MACS) on reducing the proportion of sperm with DNA fragmentation and protamine deficiency. Semen analysis and a sperm DNA fragmentation assay were performed to assess the correlations between semen parameters and the DFI in 458 semen samples. Sperm with progressive motility or non-apoptosis were isolated by DGC or MACS, respectively, in 29 normozoospermic semen samples. The effects of DGC or MACS alone and of DGC and MACS combined on reducing the amount of sperm in the sample with DNA fragmentation and protamine deficiency were investigated. The sperm DFI showed a significant correlation (r=-0.347, p<0.001) with sperm motility and morphology (r=-0.114, p<0.05) but not with other semen parameters. The DFI (11.5%±2.0%) of semen samples was significantly reduced by DGC (8.1%±4.1%) or MACS alone (7.4%±3.9%) (p<0.05). The DFI was significantly further reduced by a combination of DGC and MACS (4.1%±1.3%, p<0.05). Moreover, the combination of DGC and MACS (1.6%±1.1%, p<0.05) significantly reduced the protamine deficiency rate of semen samples compared to DGC (4.4%±3.2%) or MACS alone (3.4%±2.2%). The combination of DGC and MACS may be an effective method to isolate high-quality sperm with progressive motility, non-apoptosis, high DNA integrity, and low protamine deficiency in clinical use.

  17. Use of RNAlater in fluorescence-activated cell sorting (FACS reduces the fluorescence from GFP but not from DsRed

    Directory of Open Access Journals (Sweden)

    Epstein Miles L

    2010-12-01

    Full Text Available Abstract Background Flow cytometry utilizes signals from fluorescent markers to separate targeted cell populations for gene expression studies. However, the stress of the FACS process could change normal gene expression profiles. RNAlater could be used to stop such changes in original gene expression profiles through its ability to denature RNase and other proteins. The normal conformational structure of fluorescent proteins must be maintained in order to fluoresce. Whether or not RNAlater would affect signals from different types of intrinsic fluorescent proteins is crucial to its use in flow cytometry; this question has not been investigated in detail. Findings To address this question, we analyzed the effect of RNAlater on fluorescence intensity of GFP, YFP, DsRed and small fluorescent molecules attached to secondary antibodies (Cy2 and Texas-Red when used in flow cytometry. FACS results were confirmed with fluorescence microscopy. Our results showed that exposure of YFP and GFP containing cells to RNAlater reduces the intensity of their fluorescence to such an extent that separation of such labeled cells is difficult if not impossible. In contrast, signals from DsRed2, Cy2 and Texas-Red were not affected by RNAlater treatment. In addition, the background fluorescence and clumping of dissociated cells are altered by RNAlater treatment. Conclusions When considering gene expression studies using cell sorting with RNAlater, DsRed is the fluorescent protein of choice while GFP/YFP have severe limitations because of their reduced fluorescence. It is necessary to examine the effects of RNAlater on signals from fluorescent markers and the physical properties (e.g., clumping of the cells before considering its use in cell sorting.

  18. Magnetic cell sorting and flow cytometry sorting methods for the isolation and function analysis of mouse CD4+ CD25+ Treg cells*

    OpenAIRE

    2009-01-01

    Objective: In this paper we compared the two methods of cell sorting (magnetic cell sorting and flow cytometry sorting) for the isolation and function analysis of mouse CD4+ CD25+ regulatory T (Treg) cells, in order to inform further studies in Treg cell function. Methods: We separately used magnetic cell sorting and flow cytometry sorting to identify CD4+ CD25+ Treg cells. After magnetic cell separation, we further used flow cytometry to analyze the purity of CD4+ CD25+ Treg cells, trypan bl...

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

    Science.gov (United States)

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

    2016-09-01

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

  20. Sorting Recycled Trash: An Activity for Earth Day 2007

    Science.gov (United States)

    Harris, Mary E.; Harris, Harold H.

    2007-01-01

    Middle or high school students celebrate Earth Day on April 22, 2007 by participating in the activity to separate commingled recyclable trash to simulate sorting in a recycling center. Students would gain an appreciation for recyclable trash, after it is taken to a recycling center and learn about properties of recyclables.

  1. Unravelling the pivotal role of Alix in MVB sorting and silencing of the activated EGFR.

    Science.gov (United States)

    Sun, Sheng; Zhou, Xi; Zhang, Wei; Gallick, Gary E; Kuang, Jian

    2015-03-15

    Endosomal sorting complex required for transport (ESCRT)-III-mediated membrane invagination and scission are a critical step in multivesicular body (MVB) sorting of ubiquitinated membrane receptors, and generally thought to be required for degradation of these receptors in lysosomes. The adaptor protein Alix is critically involved in multiple ESCRT-III-mediated, membrane-remodelling processes in mammalian cells. However, Alix knockdown does not inhibit degradation of the activated epidermal growth factor receptor (EGFR) in mammalian cell lines, leading to a widely held notion that Alix is not critically involved in MVB sorting of ubiquitinated membrane receptors in mammalian cells. In the present study, we demonstrate that, despite its non-essential role in degradation of the activated EGFR, Alix plays a critical role in its MVB sorting and silencing Epidermal growth factor (EGF) stimulation of mammalian cell lines induces Alix's interaction with the ubiquitinated EGFR via the Alix V domain, and increases Alix's association with membrane-bound charged multivesicular body protein 4 (CHMP4) via the Alix Bro1 domain. Under both continuous and pulse-chase EGF stimulation conditions, inhibition of Alix's interaction with membrane-bound CHMP4, inhibition of Alix dimerization through the V domain or Alix knockdown dramatically inhibits MVB sorting of the activated EGFR and promotes sustained activation of extracellular-signal regulated kinase (ERK)1/2. Under the continuous EGF stimulation conditions, these cell treatments also retard degradation of the activated EGFR. These findings indicate that Alix is critically involved in MVB sorting of ubiquitinated membrane receptors in mammalian cells.

  2. Development of a novel cell sorting method that samples population diversity in flow cytometry.

    Science.gov (United States)

    Osborne, Geoffrey W; Andersen, Stacey B; Battye, Francis L

    2015-11-01

    Flow cytometry based electrostatic cell sorting is an important tool in the separation of cell populations. Existing instruments can sort single cells into multi-well collection plates, and keep track of cell of origin and sorted well location. However currently single sorted cell results reflect the population distribution and fail to capture the population diversity. Software was designed that implements a novel sorting approach, "Slice and Dice Sorting," that links a graphical representation of a multi-well plate to logic that ensures that single cells are sampled and sorted from all areas defined by the sort region/s. Therefore the diversity of the total population is captured, and the more frequently occurring or rarer cell types are all sampled. The sorting approach was tested computationally, and using functional cell based assays. Computationally we demonstrate that conventional single cell sorting can sample as little as 50% of the population diversity dependant on the population distribution, and that Slice and Dice sorting samples much more of the variety present within a cell population. We then show by sorting single cells into wells using the Slice and Dice sorting method that there are cells sorted using this method that would be either rarely sorted, or not sorted at all using conventional single cell sorting approaches. The present study demonstrates a novel single cell sorting method that samples much more of the population diversity than current methods. It has implications in clonal selection, stem cell sorting, single cell sequencing and any areas where population heterogeneity is of importance.

  3. Cell sorting using efficient light shaping approaches

    DEFF Research Database (Denmark)

    Banas, Andrew; Palima, Darwin; Villangca, Mark Jayson;

    2016-01-01

    distributions aimed at the positions of the detected cells. Furthermore, the beam shaping freedom provided by GPC can allow optimizations in the beam’s propagation and its interaction with the catapulted cells. © (2016) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading...... is gentler, less invasive and more economical compared to conventional FACS systems. As cells are less responsive to plastic or glass beads commonly used in the optical manipulation literature, and since laser safety would be an issue in clinical use, we develop efficient approaches in utilizing lasers...... and light modulation devices. The Generalized Phase Contrast (GPC) method that can be used for efficiently illuminating spatial light modulators or creating well-defined contiguous optical traps is supplemented by diffractive techniques capable of integrating the available light and creating 2D or 3D beam...

  4. Cell sorting using efficient light shaping approaches

    DEFF Research Database (Denmark)

    2016-01-01

    and light modulation devices. The Generalized Phase Contrast (GPC) method that can be used for efficiently illuminating spatial light modulators or creating well-defined contiguous optical traps is supplemented by diffractive techniques capable of integrating the available light and creating 2D or 3D beam...... distributions aimed at the positions of the detected cells. Furthermore, the beam shaping freedom provided by GPC can allow optimizations in the beam’s propagation and its interaction with the catapulted cells. © (2016) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading...

  5. Lipid polarity and sorting in epithelial cells

    NARCIS (Netherlands)

    van Meer, G.; Simons, K.

    1988-01-01

    Apical and basolateral membrane domains of epithelial cell plasma membranes possess unique lipid compositions. The tight junction, the structure separating the two domains, forms a diffusion barrier for membrane components and thereby prevents intermixing of the two sets of lipids. The barrier appar

  6. Large area magnetic micropallet arrays for cell colony sorting.

    Science.gov (United States)

    Cox-Muranami, Wesley A; Nelson, Edward L; Li, G P; Bachman, Mark

    2016-01-01

    A new micropallet array platform for adherent cell colony sorting has been developed. The platform consisted of thousands of square plastic pallets, 270 μm by 270 μm on each side, large enough to hold a single colony of cells. Each pallet included a magnetic core, allowing them to be collected with a magnet after being released using a microscope mounted laser system. The micropallets were patterned from 1002F epoxy resist and were fabricated on translucent, gold coated microscope slides. The gold layer was used as seed for electroplating the ferromagnetic cores within every individual pallet. The gold layer also facilitated the release of each micropallet during laser release. This array allows for individual observation, sorting and collection of isolated cell colonies for biological cell colony research. In addition to consistent release and recovery of individual colonies, we demonstrated stable biocompatibility and minimal loss in imaging quality compared to previously developed micropallet arrays.

  7. Purification of Definitive Endoderm Generated from Pluripotent Stem Cells by Magnetic Cell Sorting.

    Science.gov (United States)

    Diekmann, Ulf; Davenport, Claudia; Kresse, Jasmin; Naujok, Ortwin

    2017-02-02

    Pluripotent stem cells have the capability to differentiate into any somatic cell type of the human body. The generation of surrogate cells for the treatment of liver, lung, and pancreatic diseases is of great medical interest. First, the in vitro formation into cells of the definitive endoderm is required. Upon commitment into this lineage, the cells express transcription factors such as FOXA2, SOX17, HNF1B; GATA family members; and the surface protein CXCR4. Unfortunately, some pluripotent stem cells resist the differentiation and contaminate the culture. Thus, we describe here an endoderm differentiation protocol, which yields endoderm-committed cells in high numbers in a 4-day treatment protocol. Second, a method for the purification of CXCR4-positive endoderm cells by magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS) is described. The purification by MACS is quick and reliable and can be used to obtain pure endoderm cells either meant for downstream analysis such as omics or further differentiation experiments into endoderm-derived somatic cells. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  8. Numerical Model of Streaming DEP for Stem Cell Sorting

    Directory of Open Access Journals (Sweden)

    Rucha Natu

    2016-11-01

    Full Text Available Neural stem cells are of special interest due to their potential in neurogenesis to treat spinal cord injuries and other nervous disorders. Flow cytometry, a common technique used for cell sorting, is limited due to the lack of antigens and labels that are specific enough to stem cells of interest. Dielectrophoresis (DEP is a label-free separation technique that has been recently demonstrated for the enrichment of neural stem/progenitor cells. Here we use numerical simulation to investigate the use of streaming DEP for the continuous sorting of neural stem/progenitor cells. Streaming DEP refers to the focusing of cells into streams by equilibrating the dielectrophoresis and drag forces acting on them. The width of the stream should be maximized to increase throughput while the separation between streams must be widened to increase efficiency during retrieval. The aim is to understand how device geometry and experimental variables affect the throughput and efficiency of continuous sorting of SC27 stem cells, a neurogenic progenitor, from SC23 cells, an astrogenic progenitor. We define efficiency as the ratio between the number of SC27 cells over total number of cells retrieved in the streams, and throughput as the number of SC27 cells retrieved in the streams compared to their total number introduced to the device. The use of cylindrical electrodes as tall as the channel yields streams featuring >98% of SC27 cells and width up to 80 µm when using a flow rate of 10 µL/min and sample cell concentration up to 105 cells/mL.

  9. Psychometric properties of the Arab Heritage Activity Card Sort.

    Science.gov (United States)

    Hamed, Razan; Holm, Margo B

    2013-03-01

    The Activity Card Sort is a valid and reliable assessment tool that was created to assess Participation. It has been translated to several languages and adapted to different international cultures. The most recent version of this tool is the Arabic Heritage Activity Card Sort (A-ACS). The purpose of this study was to establish the psychometric properties of the new Arabic version in Jordanian adults. Forty three Jordanian patients with multiple sclerosis (MS) and 62 healthy adults were recruited to test the psychometric properties of the tool. The A-ACS correlated moderately with the participation index of the Mayo-Portland Adaptability Inventory (r = -0.458, p Heritage of the Activity Card Sort is a valid and reliable tool for Arabic-speaking occupational therapists to use when assessing participation in Jordanian patients with MS or healthy adults. Limitations of this study include using only one diagnostic group from Jordan and examining only the Recovery and Community Versions of the tool. Future studies are needed to examine further psychometric properties for patients with different diagnoses and from different countries in the Arabic region for all three versions of the A-ACS.

  10. Design of Slit between Micro Cylindrical Pillars for Cell Sorting

    Directory of Open Access Journals (Sweden)

    Yusuke Takahashi

    2016-12-01

    Full Text Available Micro slits have been designed between micro cylindrical pillars to sort biological cells. Micro cylindrical pillars of 0.02 mm diameter and 0.055 mm height were fabricated on the glass plate using the photolithography technique. Variation was made on the gap between pillars: 0.01 mm, 0.02 mm, 0.03 mm, and 0.04 mm. The micro pillars are set in the flow path between parallel plates, of which dimension of the cross section is 5 mm width and 0.055 mm height. Three kinds of biological cells were used in the test alternatively: C2C12 (mouse myoblast cell line originated with cross-striated muscle of C3H mouse, Hepa1-6 (mouse hepatoma cell line of C57L mouse, or swine red blood cell. The suspension of cells was introduced into the slits by the syringe pump at the flow rate between 0.4 and 500 cm3/hour. The deformation of the cell at the slit can be observed by the microscope. The experimental results show that the designed slit has capability for sorting cells according to the size and deformability of the cell.

  11. Improved method for bacterial cell capture after flow cytometry cell sorting.

    Science.gov (United States)

    Guillebault, D; Laghdass, M; Catala, P; Obernosterer, I; Lebaron, P

    2010-11-01

    Fixed cells with different nucleic acid contents and scatter properties (low nucleic acid [LNA], high nucleic acid 1 [HNA1], and HNA2) were sorted by flow cytometry (FCM). For each sort, 10,000 cells were efficiently captured on poly-l-lysine-coated microplates, resulting in efficient and reproducible PCR amplification.

  12. A kinetic mechanism for cell sorting based on local variations in cell motility

    OpenAIRE

    Strandkvist, Charlotte; Juul, Jeppe; Baum, Buzz; Kabla, Alexandre J.; Duke, Tom

    2014-01-01

    Our current understanding of cell sorting relies on physical difference, either in the interfacial properties or motile force, between cell types. But is such asymmetry a prerequisite for cell sorting? We test this using a minimal model in which the two cell populations are identical with respect to their physical properties and differences in motility arise solely from how cells interact with their surroundings. The model resembles the Schelling model used in social sciences to study segrega...

  13. Magnetic activated cell sorting and its application in the studies of male infertility%磁性活性细胞分选技术在男性不育研究中的应用

    Institute of Scientific and Technical Information of China (English)

    邓雪连

    2012-01-01

    Magnetic activated cell sorting ( MACS) is considered as a flexible, fast, specific and simple cell sorting system that can separate target cells effectively according to specific markers on the cell surface, for which it has won a wide clinical application. MACS offers a new platform for male infertility research, as well as a novel idea for applying this technology in the optimization of semen quality and the isolation of germ cells. This article briefly introduces the basic principles of MACS, and summaries its present and potential clinical application in male infertility research, as in spermatozoa selection and cryopreservation, and the isolation of spermat-ogonial stem cells and germ cells.%磁性活性细胞分选法(MACS)根据细胞表面特异的标记物,在分子水平对目的细胞进行有效分选,具有简易、快速、灵活、特异性高的特点,在临床方面有着广泛的应用.MACS也为男性不育提供了一个新的研究平台,将MACS用于精液质量优化与生殖细胞分离是男性不育研究的一个新思路.本文简要介绍了MACS的基本原理,综述了MACS在精子优选、冷冻保存、精原干细胞及生精细胞分离等男性不育研究方面的应用现状和临床应用前景.

  14. Fast polyhedral cell sorting for interactive rendering of unstructured grids

    Energy Technology Data Exchange (ETDEWEB)

    Combra, J; Klosowski, J T; Max, N; Silva, C T; Williams, P L

    1998-10-30

    Direct volume rendering based on projective methods works by projecting, in visibility order, the polyhedral cells of a mesh onto the image plane, and incrementally compositing the cell's color and opacity into the final image. Crucial to this method is the computation of a visibility ordering of the cells. If the mesh is ''well-behaved'' (acyclic and convex), then the MPVO method of Williams provides a very fast sorting algorithm; however, this method only computes an approximate ordering in general datasets, resulting in visual artifacts when rendered. A recent method of Silva et al. removed the assumption that the mesh is convex, by means of a sweep algorithm used in conjunction with the MPVO method; their algorithm is substantially faster than previous exact methods for general meshes. In this paper we propose a new technique, which we call BSP-XMPVO, which is based on a fast and simple way of using binary space partitions on the boundary elements of the mesh to augment the ordering produced by MPVO. Our results are shown to be orders of magnitude better than previous exact methods of sorting cells.

  15. Separation and sorting of cells in microsystems using physical principles

    Science.gov (United States)

    Lee, Gi-Hun; Kim, Sung-Hwan; Ahn, Kihoon; Lee, Sang-Hoon; Park, Joong Yull

    2016-01-01

    In the last decade, microfabrication techniques have been combined with microfluidics and applied to cell biology. Utilizing such new techniques, various cell studies have been performed for the research of stem cells, immune cells, cancer, neurons, etc. Among the various biological applications of microtechnology-based platforms, cell separation technology has been highly regarded in biological and clinical fields for sorting different types of cells, finding circulating tumor cells (CTCs), and blood cell separation, amongst other things. Many cell separation methods have been created using various physical principles. Representatively, these include hydrodynamic, acoustic, dielectrophoretic, magnetic, optical, and filtering methods. In this review, each of these methods will be introduced, and their physical principles and sample applications described. Each physical principle has its own advantages and disadvantages. The engineers who design the systems and the biologists who use them should understand the pros and cons of each method or principle, to broaden the use of microsystems for cell separation. Continuous development of microsystems for cell separation will lead to new opportunities for diagnosing CTCs and cancer metastasis, as well as other elements in the bloodstream.

  16. 免疫磁珠法筛选人乳牙牙髓干细胞及其培养鉴定%Isolation and identification of stem cells derived from human exfoliated deciduous teeth by magnetic activated cell sorting

    Institute of Scientific and Technical Information of China (English)

    丁祥龙; 陈柯; 申元源

    2011-01-01

    目的 应用免疫磁珠法分离筛选人乳牙牙髓干细胞,并做培养鉴定.方法 采用STRO-1为标记物以免疫磁珠分离筛选人乳牙牙髓干细胞,观察细胞形态及生长情况,流式细胞仪检测细胞表型,并检测细胞体外多向分化能力.结果 应用免疫磁珠法筛选人乳牙牙髓细胞可获得乳牙牙髓于细胞,经统计学处理证明看其生长速度慢于牙髓细胞,细胞表型分析证实CD29、CD105高表达,CD34、CD45低表达.矿化诱导和成脂诱导证实STRO-1+乳牙牙髓细胞具有干细胞多向分化的能力.结论 免疫磁珠法是有效的分离纯化人乳牙牙髓干细胞的方法.所分离的细胞具有干细胞的表型特点及多向分化能力.%Objective To isolate stem cells from human exfoliated deciduous teeth (SHEDs) and identify their phenotypes and multi-lineage differentiation potential. Methods Human pulp tissue from exfoliated deciduous teeth were dissected and digested to obtain the single cell suspension. The SHEDs selected by magnetic activated cell sorting system (MACS) were identified by examination of the cell morphology and growth in vitro and detection of the expressions of the cell markers. Osteogenic and adipogenic induction was performed to test the multi-lineage differentiation potential of the cells. Results SHEDs were successfully isolated from human exfoliated deciduous teeth. SHEDs showed a lower growth rate than dental pulp cells and displayed high expressions of CD29 and CD105 but low expressions of CD34 and CD45 as shown by flow cytometry. Experiments of in vitro induction demonstrated a strong potential of the STRO-1+ SHEDs for osteogenic and adipogenic differentiation. Conclusion Immunomagnetic bead selection can be used to isolate and purify SHEDs, and the STRO-1+ SHEDs show the characteristics of stem cells with multipotent differentiation potentials.

  17. Adhesion functions in cell sorting by mechanically coupling the cortices of adhering cells.

    Science.gov (United States)

    Maître, Jean-Léon; Berthoumieux, Hélène; Krens, Simon Frederik Gabriel; Salbreux, Guillaume; Jülicher, Frank; Paluch, Ewa; Heisenberg, Carl-Philipp

    2012-10-12

    Differential cell adhesion and cortex tension are thought to drive cell sorting by controlling cell-cell contact formation. Here, we show that cell adhesion and cortex tension have different mechanical functions in controlling progenitor cell-cell contact formation and sorting during zebrafish gastrulation. Cortex tension controls cell-cell contact expansion by modulating interfacial tension at the contact. By contrast, adhesion has little direct function in contact expansion, but instead is needed to mechanically couple the cortices of adhering cells at their contacts, allowing cortex tension to control contact expansion. The coupling function of adhesion is mediated by E-cadherin and limited by the mechanical anchoring of E-cadherin to the cortex. Thus, cell adhesion provides the mechanical scaffold for cell cortex tension to drive cell sorting during gastrulation.

  18. Protein characterization of intracellular target-sorted, formalin-fixed cell subpopulations

    Science.gov (United States)

    Sadick, Jessica S.; Boutin, Molly E.; Hoffman-Kim, Diane; Darling, Eric M.

    2016-01-01

    Cellular heterogeneity is inherent in most human tissues, making the investigation of specific cell types challenging. Here, we describe a novel, fixation/intracellular target-based sorting and protein extraction method to provide accurate protein characterization for cell subpopulations. Validation and feasibility tests were conducted using homogeneous, neural cell lines and heterogeneous, rat brain cells, respectively. Intracellular proteins of interest were labeled with fluorescent antibodies for fluorescence-activated cell sorting. Reproducible protein extraction from fresh and fixed samples required lysis buffer with high concentrations of Tris-HCl and sodium dodecyl sulfate as well as exposure to high heat. No deterioration in protein amount or quality was observed for fixed, sorted samples. For the feasibility experiment, a primary rat subpopulation of neuronal cells was selected for based on high, intracellular β-III tubulin signal. These cells showed distinct protein expression differences from the unsorted population for specific (phosphorylated tau) and non-specific (total tau) protein targets. Our approach allows for determining more accurate protein profiles directly from cell types of interest and provides a platform technology in which any cell subpopulation can be biochemically investigated. PMID:27666089

  19. Novel serial positive enrichment technology enables clinical multiparameter cell sorting.

    Directory of Open Access Journals (Sweden)

    Christian Stemberger

    Full Text Available A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve--especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4(high/CD25(high/CD45RA(high 'regulatory T cells' and CD8(high/CD62L(high/CD45RA(neg 'central memory T cells', have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research.

  20. Cell sorting enriches Escherichia coli mutants that rely on peptidoglycan endopeptidases to suppress highly aberrant morphologies.

    Science.gov (United States)

    Laubacher, Mary E; Melquist, Amy L; Chandramohan, Lakshmi; Young, Kevin D

    2013-02-01

    Bacterial morphology imparts physiological advantages to cells in different environments and, judging by the fidelity with which shape is passed to daughter cells, is a tightly regulated characteristic. Surprisingly, only in the past 10 to 15 years has significant headway been made in identifying the mechanisms by which cells create and maintain particular shapes. One reason for this is that the relevant discoveries have relied heavily on the arduous, somewhat subjective process of manual microscopy. Here, we show that flow cytometry, coupled with the sorting capability of fluorescence-activated cell sorting (FACS), can detect, quantify, and enrich bacteria with morphological alterations. The light scattering properties of several highly aberrant morphological mutants of Escherichia coli were characterized by flow cytometry. Cells from a region that overlapped the distribution of normal rod-shaped cells were collected by FACS and reincubated. After 4 to 15 iterations of this enrichment process, suppressor mutants were isolated that returned almost all the population to a near-normal shape. Suppressors were successfully isolated from strains lacking three or four penicillin binding proteins (PBPs) but not from a mutant lacking a total of seven PBPs. The peptidoglycan endopeptidase, AmpH, was identified as being important for the suppression process, as was a related endopeptidase, MepA. The results validate the use of cell sorting as a means for studying bacterial morphology and identify at least one new class of enzymes required for the suppression of cell shape defects.

  1. Micro and nanofluidic structures for cell sorting and genomic analysis

    Science.gov (United States)

    Morton, Keith J.

    Microfluidic systems promise rapid analysis of small samples in a compact and inexpensive format. But direct scaling of lab bench protocols on-chip is challenging because laminar flows in typical microfluidic devices are characterized by non-mixing streamlines. Common microfluidic mixers and sorters work by diffusion, limiting application to objects that diffuse slowly such as cells and DNA. Recently Huang et.al. developed a passive microfluidic element to continuously separate bio-particles deterministically. In Deterministic Lateral Displacement (DLD), objects are sorted by size as they transit an asymmetric array of microfabricated posts. This thesis further develops DLD arrays with applications in three broad new areas. First the arrays are used, not simply to sort particles, but to move streams of cells through functional flows for chemical treatment---such as on-chip immunofluorescent labeling of blood cells with washing, and on-chip E.coli cell lysis with simultaneous chromosome extraction. Secondly, modular tiling of the basic DLD element is used to construct complex particle handling modes that include beam steering for jets of cells and beads. Thirdly, nanostructured DLD arrays are built using Nanoimprint Lithography (NIL) and continuous-flow separation of 100 nm and 200 nm size particles is demonstrated. Finally a number of ancillary nanofabrication techniques were developed in support of these overall goals, including methods to interface nanofluidic structures with standard microfluidic components such as inlet channels and reservoirs, precision etching of ultra-high aspect ratio (>50:1) silicon nanostructures, and fabrication of narrow (˜ 35 nm) channels used to stretch genomic length DNA.

  2. Finite-size corrections to scaling behavior in sorted cell aggregates.

    Science.gov (United States)

    Klopper, A V; Krens, G; Grill, S W; Heisenberg, C-P

    2010-10-01

    Cell sorting is a widespread phenomenon pivotal to the early development of multicellular organisms. In vitro cell sorting studies have been instrumental in revealing the cellular properties driving this process. However, these studies have as yet been limited to two-dimensional analysis of three-dimensional cell sorting events. Here we describe a method to record the sorting of primary zebrafish ectoderm and mesoderm germ layer progenitor cells in three dimensions over time, and quantitatively analyze their sorting behavior using an order parameter related to heterotypic interface length. We investigate the cell population size dependence of sorted aggregates and find that the germ layer progenitor cells engulfed in the final configuration display a relationship between total interfacial length and system size according to a simple geometrical argument, subject to a finite-size effect.

  3. Viable cell sorting of dinoflagellates by multi-parametric flow cytometry.

    Science.gov (United States)

    Electronic cell sorting for isolation and culture of dinoflagellates and other marine eukaryotic phytoplankton was compared to the traditional method of manually picking of cells using a micropipette. Trauma to electronically sorted cells was not a limiting factor as fragile dinoflagellates, such a...

  4. Immunomagnetic Indirect Positive Sorting of Precartilaginous Stem Cells from Neonatal Rat

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    To investigate the technique of sorting high-purity precartilaginous stem cells from rat's perichondrium, neonatal rat's perichondrium cells suspensions were incubated with monoclone antibody of anti-fibroblast growth factor receptor-3 (anti-FGFR-3), and the labeled cells were separated from the suspension in the magnetic field by immuno-beads coated with the second antibody. Purityof the sorted neural stem cells was found to be 93.0 %-99.0 %, with living cells amounting to 80 %-85 %. The magnetic cell sorting system could effectively separate precartilaginous stem cells fromperichondrium cell suspensions.

  5. Purification of microglia by two-step magnetic activated cell sorting%免疫磁珠分选两步法纯化原代小胶质细胞

    Institute of Scientific and Technical Information of China (English)

    孙珊珊; 王蓓蓓; 鞠莉莉; 曾辉; 徐群渊

    2012-01-01

    目的 神经系统感染研究需获得纯度高、细胞生物学状态接近体内的小胶质细胞.既往分离纯化方法 不能满足研究需要,需建立新的高效纯化方法.方法 获得小鼠全脑单细胞悬液后,应用磁珠分选阳选法,经过两步分选获得小胶质细胞.用流式细胞仪检测小胶质细胞的纯度,瑞氏-姬姆萨染色观察细胞形态,采用7-AAD染色鉴定分选后细胞的活性.结果 应用免疫磁珠分选一步法可以获得纯度达(59.98 ± 13.61)%的小胶质细胞;两步法进一步将细胞纯度提高至(97.62 ± 1.35)%.该方法 所获得的小胶质细胞活性良好,形态正常.结论 免疫磁珠分选两步法能够稳定地获得高纯度的小胶质细胞,对小胶质细胞的活性和形态无显著影响,可用于后续中枢神经系统急性感染的体外研究.%Objective The study on nervous system infection requires us to obtain microglia with high purity and biological status. However, present methods for microglia purification can not meet these requirements. It is necessary to establish an efficient separation method. Methods After having prepared the whole mouse brain cell suspension, two-step magnetic activated cell positive sorting ( MACS ) technique was applied specifically for CDllb+ microglia sorting. The purity of the cells was detected by flow cytometry, the morphology of the cells was observed by Wright-Giemsa staining, and the viability of the cells were determined by 7-AAD staining. Results The study obtained mice microglia with purity of ( 59. 98 ± 13.61 )% for Step 1 MACS and ( 97. 62 ± 1. 35 )% for Step 2 MACS, which could bear good viability and morphology. Conclusions The two-step magnetic cell sorting technique could obtain high purity microglia stably, with no significant effect on its activity and morphology. The method suits for studying the infection in CNS.

  6. Lgr5 positive stem cells sorted from small intestines of diabetic mice differentiate into higher proportion of absorptive cells and Paneth cells in vitro.

    Science.gov (United States)

    Zhong, Xian-Yang; Yu, Tao; Zhong, Wa; Li, Jie-Yao; Xia, Zhong-Sheng; Yuan, Yu-Hong; Yu, Zhong; Chen, Qi-Kui

    2015-08-01

    Intestinal epithelial stem cells (IESCs) can differentiate into all types of intestinal epithelial cells (IECs) and Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) is a marker for IESC. Previous studies reported enhanced proliferation of IECs in diabetic mice. In this study, the in vitro differentiation of Lgr5 positive IESCs sorted from diabetic mice was further investigated. The diabetic mouse model was induced by streptozotocin (STZ), and crypt IECs were isolated from small intestines. Subsequently, Lgr5 positive IESCs were detected by flow cytometry (FCM) and sorted by magnetic activated cell sorting (MACS). Differentiation of the sorted IESCs was investigated by detecting the IEC markers in the diabetic mice using immunostaining, quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR), and Western blot analysis, which was compared with normal mice. We found that the proportion of Lgr5 positive cells in the crypt IECs of diabetic mice was higher than that of control mice (P absorptive cell marker sucrase-isomaltase (SI) and the Paneth cell marker lysozyme 1 (Lyz1) were more highly expressed in the differentiated cells derived from Lgr5 positive IESCs of diabetic mice in vitro (P small intestines of STZ-induced diabetic mice. Lgr5 positive IESCs sorted from the diabetic mice can differentiate into a higher proportion of absorptive cells and Paneth cells in vitro. We characterized the expression of Lgr5 in the small intestine of diabetic mice, and sorted Lgr5 positive intestinal epithelial stem cells (IESCs) for investigating their differentiation in vitro. We proved that the quantity of Lgr5 positive IESCs was significantly increased in the small intestines of diabetic mice. IESCs sorted from the diabetic mice can differentiate into a higher proportion of absorptive cells and Paneth cells in vitro.

  7. A method for high purity sorting of rare cell subsets applied to TDC.

    Science.gov (United States)

    Kuka, Mirela; Ashwell, Jonathan D

    2013-12-31

    T(DC) are a recently described subset of polyclonal αβ T-cells with dendritic cell properties. Because of their low number in peripheral immune compartments, isolation and characterization of T(DC) with existing purification methods are technically challenging. Here we describe a customized gating strategy and a flow cytometry-based cell sorting protocol for isolation of T(DC). The protocol was developed because, despite very conservative gating for dead-cell and doublet exclusion, cells obtained with normal sorting procedures were enriched for T(DC) but not pure. Re-sorting the output of the first round of sorting results in highly pure T(DC). Cells obtained with this method are viable and can be used for in vitro characterization. Moreover, this double-round sorting strategy can be universally applied to the isolation of other rare cell subsets.

  8. Sorting live stem cells based on Sox2 mRNA expression.

    Directory of Open Access Journals (Sweden)

    Hans M Larsson

    Full Text Available While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES and neural stem cells (NSC. One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB(+SSEA1(+ cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB(+ cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB(- cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner.

  9. Major proteins in normal human lymphocyte subpopulations separated by fluorescence-activated cell sorting and analyzed by two-dimensional gel electrophoresis

    DEFF Research Database (Denmark)

    Madsen, P S; Hokland, M; Ellegaard, J

    1988-01-01

    We have compared the overall patterns of protein synthesis of normal human lymphocyte subpopulations taken from five volunteers using high resolution two-dimensional gel electrophoresis. The lymphocytes were isolated using density gradient centrifugation, labeled with subtype-specific Mo......Abs, and separated to a high degree of homogeneity by FACS into CD4+ helper T cells, CD8+ suppressor T cells, CD20+ B cells, and N901 (NHK-1)+ NK cells. The four lymphocyte subpopulations were labeled with [35S]methionine for 14 hr, solubilized in lysis buffer, and analyzed by two-dimensional gel electrophoresis...

  10. Advanced real-time classification methods for flow cytometry data analysis and cell sorting

    Science.gov (United States)

    Leary, James F.; Reece, Lisa M.; Hokanson, James A.; Rosenblatt, Judah I.

    2002-05-01

    While many flow cytometric data analysis and 'discovery' methods have been developed, few of these have been applied to the problem of separating out purified cell subpopulations by cell sorting. The fundamental problem is that the data analysis techniques have been performed using relatively slow computational methods that take far more time than is allowed by the sort decision on a cell sorter (typically less than a millisecond). Thus cell sorting, which is really a form of 'real-time data classification,' is usually done with few, if any, multivariate statistical tools used either in the sort decision or in the evaluation of the correctness of the classification. We have developed new multivariate data analysis and 'data discovery' methods that can be implemented for real-time data classification for cell sorting using linked lookup tables. One multivariate 'data discovery' method, 'subtractive clustering,' has been used to find which clusters of cells are different between two or more files (cell samples) and to help guide analysis or sort boundaries for these cell subpopulations. Multivariate statistical methods (e.g. principal component analysis or discriminant function analysis) were implemented in linked lookup tables to establish analysis/sort boundaries that include 'costs (or penalties) of misclassification. Costs of misclassification provided a measure of the quality of the analysis/sort boundary and were expressed in simple terms that describe the tradeoff between yield and purity.

  11. Kinematics Card Sort Activity: Insight into Students' Thinking

    Science.gov (United States)

    Berryhill, Erin; Herrington, Deborah; Oliver, Keith

    2016-12-01

    Kinematics is a topic students are unknowingly aware of well before entering the physics classroom. Students observe motion on a daily basis. They are constantly interpreting and making sense of their observations, unintentionally building their own understanding of kinematics before receiving any formal instruction. Unfortunately, when students take their prior conceptions to understand a new situation, they often do so in a way that inaccurately connects their learning. We were motivated to identify strategies to help our students make accurate connections to their prior knowledge and understand kinematics at a deeper level. To do this, we integrated a formative assessment card sort into a kinematic graphing unit within an introductory high school physics course. Throughout the activities, we required students to document and reflect upon their thinking. This allowed their learning to build upon their own previously held conceptual understanding, which provided an avenue for cognitive growth. By taking a more direct approach to eliciting student reasoning, we hoped to improve student learning and guide our assessment of their learning.

  12. The Psychometric Properties of the Arabic Preschool Activity Card Sort

    Directory of Open Access Journals (Sweden)

    Somaya H. Malkawi

    2017-01-01

    Full Text Available Background. The Preschool Activity Card Sort (PACS is an interview-based assessment tool to measure participation of preschool children with age range from 3 to 6 years. Objective of Study. The purpose of this study was to establish the psychometric properties of the recently translated Arabic PACS (A-PACS. Methods. One hundred fifty-one Jordanian parents participated in the study representing different geographical areas. Children were almost equally distributed between males and females and into three age groups. Construct and concurrent validity were examined as well as the internal consistency of the scale and the test-retest reliability. Findings. The A-PACS was able to differentiate between the participation level of young and old children in the domains of education, community mobility, and low demand leisure of the A-PACS giving evidence to its construct validity and it significantly correlated with some aspects of the Vineland Adaptive Behavior Scale (VABS giving evidence to its concurrent validity. The A-PACS showed excellent overall internal consistency (α=.859 for all domains and good test-retest reliability (r=.976, p<.001. Conclusion. The A-PACS can be considered as a valid and reliable tool to measure participation of preschool children with normal development from Arabic cultures. Future studies should focus on the validity of the A-PACS for use with children with disabilities.

  13. The Psychometric Properties of the Arabic Preschool Activity Card Sort

    Science.gov (United States)

    Abu-Dahab, Sana M. N.; Amro, Ahmad F.; Almasri, Nihad A.

    2017-01-01

    Background The Preschool Activity Card Sort (PACS) is an interview-based assessment tool to measure participation of preschool children with age range from 3 to 6 years. Objective of Study The purpose of this study was to establish the psychometric properties of the recently translated Arabic PACS (A-PACS). Methods One hundred fifty-one Jordanian parents participated in the study representing different geographical areas. Children were almost equally distributed between males and females and into three age groups. Construct and concurrent validity were examined as well as the internal consistency of the scale and the test-retest reliability. Findings The A-PACS was able to differentiate between the participation level of young and old children in the domains of education, community mobility, and low demand leisure of the A-PACS giving evidence to its construct validity and it significantly correlated with some aspects of the Vineland Adaptive Behavior Scale (VABS) giving evidence to its concurrent validity. The A-PACS showed excellent overall internal consistency (α = .859) for all domains and good test-retest reliability (r = .976, p < .001). Conclusion The A-PACS can be considered as a valid and reliable tool to measure participation of preschool children with normal development from Arabic cultures. Future studies should focus on the validity of the A-PACS for use with children with disabilities.

  14. A cell sorting and trapping microfluidic device with an interdigital channel

    Science.gov (United States)

    Tu, Jing; Qiao, Yi; Xu, Minghua; Li, Junji; Liang, Fupeng; Duan, Mengqin; Ju, An; Lu, Zuhong

    2016-12-01

    The growing interest in cell sorting and trapping is driving the demand for high performance technologies. Using labeling techniques or external forces, cells can be identified by a series of methods. However, all of these methods require complicated systems with expensive devices. Based on inherent differences in cellular morphology, cells can be sorted by specific structures in microfluidic devices. The weir filter is a basic and efficient cell sorting and trapping structure. However, in some existing weir devices, because of cell deformability and high flow velocity in gaps, trapped cells may become stuck or even pass through the gaps. Here, we designed and fabricated a microfluidic device with interdigital channels for cell sorting and trapping. The chip consisted of a sheet of silicone elastomer polydimethylsiloxane and a sheet of glass. A square-wave-like weir was designed in the middle of the channel, comprising the interdigital channels. The square-wave pattern extended the weir length by three times with the channel width remaining constant. Compared with a straight weir, this structure exhibited a notably higher trapping capacity. Interdigital channels provided more space to slow down the rate of the pressure decrease, which prevented the cells from becoming stuck in the gaps. Sorting a mixture K562 and blood cells to trap cells demonstrated the efficiency of the chip with the interdigital channel to sort and trap large and less deformable cells. With stable and efficient cell sorting and trapping abilities, the chip with an interdigital channel may be widely applied in scientific research fields.

  15. Side population sorting separates subfractions of cycling and non-cycling intestinal stem cells

    Directory of Open Access Journals (Sweden)

    Richard J. von Furstenberg

    2014-03-01

    Full Text Available We report here that side population (SP sorting allows for the simultaneous isolation of two intestinal stem cell (ISC subsets from wild-type (WT mice which are phenotypically different and represent cycling and non-cycling pools of cells. Following 5-ethynyl-2′-deoxyuridine (EdU injection, in the upper side population (USP the percentage of EdU+ was 36% showing this fraction to be highly proliferative. In the lower side population (LSP, only 0.4% of cells were EdU+, indicating this fraction to be predominantly non-cycling. Using Lgr5-EGFP mice, we show that Lgr5-EGFPhi cells, representing actively cycling ISCs, are essentially exclusive to the USP. In contrast, using histone 2B-YFP mice, SP analysis revealed YFP label retaining cells (LRCs in both the USP and the LSP. Correspondingly, evaluation of the SP fractions for mRNA markers by qRT-PCR showed that the USP was enriched in transcripts associated with both quiescent and active ISCs. In contrast, the LSP expressed mRNA markers of quiescent ISCs while being de-enriched for those of the active ISC. Both the USP and LSP are capable of generating enteroids in culture which include the four intestinal lineages. We conclude that sorting of USP and LSP fractions represents a novel isolation of cycling and non-cycling ISCs from WT mice.

  16. SEPARATION OF PERIPORTAL AND PERIVENOUS RAT HEPATOCYTES BY FLUORESCENCE-ACTIVATED CELL SORTING - CONFIRMATION WITH COLLOIDAL GOLD AS AN EXOGENOUS MARKER

    NARCIS (Netherlands)

    BRAAKMAN, [No Value; KEIJ, J; HARDONK, MJ; MEIJER, DKF; GROOTHUIS, GMM

    1991-01-01

    Periportal and perivenous hepatocytes are known to display various functional differences. In this study we present a new method to separate periportal and perivenous cells: after selectively loading zone 1 or zone 3 with the fluorescent label acridine orange in an antegrade or retrograde perfusion,

  17. Analysis of planktonic community structure and trophic interactions using refined isotopic signatures determined by combining fluorescence-activated cell sorting and isotope-ratio mass spectrometry

    NARCIS (Netherlands)

    Pel, R.; Floris, V.; Hoogveld, H.L.

    2004-01-01

    1. Thermally assisted hydrolysis and methylation of cellular lipids, by means of Curie-point pyrolysis of intact whole cells in the presence of a quaternary ammonium hydroxide reagent, provided analytical access (pyrolysis-gas chromatography; Py-GC) to the very small amounts of algal carbon delivere

  18. Cell Specific eQTL Analysis without Sorting Cells.

    Directory of Open Access Journals (Sweden)

    Harm-Jan Westra

    2015-05-01

    Full Text Available The functional consequences of trait associated SNPs are often investigated using expression quantitative trait locus (eQTL mapping. While trait-associated variants may operate in a cell-type specific manner, eQTL datasets for such cell-types may not always be available. We performed a genome-environment interaction (GxE meta-analysis on data from 5,683 samples to infer the cell type specificity of whole blood cis-eQTLs. We demonstrate that this method is able to predict neutrophil and lymphocyte specific cis-eQTLs and replicate these predictions in independent cell-type specific datasets. Finally, we show that SNPs associated with Crohn's disease preferentially affect gene expression within neutrophils, including the archetypal NOD2 locus.

  19. An IB-LBM study of continuous cell sorting in deterministic lateral displacement arrays

    Science.gov (United States)

    Wei, Qiang; Xu, Yuan-Qing; Tang, Xiao-Ying; Tian, Fang-Bao

    2016-12-01

    The deterministic lateral displacement (DLD) is an important method used to sort particles and cells of different sizes. In this paper, the flexible cell sorting with the DLD method is studied by using a numerical model based on the immersed boundary-lattice Boltzmann method (IB-LBM). In this model, the fluid motion is solved by the LBM, and the cell membrane-fluid interaction is modeled with the LBM. The proposed model is validated by simulating the rigid particle sorted with the DLD method, and the results are found in good agreement with those measured in experiments. We first study the effect of flexibility on a single cell and multiple cells continuously going through a DLD device. It is found that the cell flexibility can significantly affect the cell path, which means the flexibility could have significant effects on the continuous cell sorting by the DLD method. The sorting characteristics of white blood cells and red blood cells are further studied by varying the spatial distribution of cylinder arrays and the initial cell-cell distance. The numerical results indicate that a well concentrated cell sorting can be obtained under a proper arrangement of cylinder arrays and a large enough initial cell-cell distance.

  20. Moving forward moving backward: directional sorting of chemotactic cells due to size and adhesion differences.

    Directory of Open Access Journals (Sweden)

    Jos Käfer

    2006-06-01

    Full Text Available Differential movement of individual cells within tissues is an important yet poorly understood process in biological development. Here we present a computational study of cell sorting caused by a combination of cell adhesion and chemotaxis, where we assume that all cells respond equally to the chemotactic signal. To capture in our model mesoscopic properties of biological cells, such as their size and deformability, we use the Cellular Potts Model, a multiscale, cell-based Monte Carlo model. We demonstrate a rich array of cell-sorting phenomena, which depend on a combination of mescoscopic cell properties and tissue level constraints. Under the conditions studied, cell sorting is a fast process, which scales linearly with tissue size. We demonstrate the occurrence of "absolute negative mobility", which means that cells may move in the direction opposite to the applied force (here chemotaxis. Moreover, during the sorting, cells may even reverse the direction of motion. Another interesting phenomenon is "minority sorting", where the direction of movement does not depend on cell type, but on the frequency of the cell type in the tissue. A special case is the cAMP-wave-driven chemotaxis of Dictyostelium cells, which generates pressure waves that guide the sorting. The mechanisms we describe can easily be overlooked in studies of differential cell movement, hence certain experimental observations may be misinterpreted.

  1. Human Periodontal Ligament Derived Progenitor Cells: Effect of STRO-1 Cell Sorting and Wnt3a Treatment on Cell Behavior

    Directory of Open Access Journals (Sweden)

    Xiang-Zhen Yan

    2014-01-01

    Full Text Available Objectives. STRO-1 positive periodontal ligament cells (PDLCs and unsorted PDLCs have demonstrated potential for periodontal regeneration, but the comparison between unsorted cells and the expanded STRO-1 sorted cells has never been reported. Additionally, Wnt3a is involved in cell proliferation thus may benefit in vitro PDLC expansion. The aim was to evaluate the effect of STRO-1 cell sorting and Wnt3a treatment on cell behavior of human PDLCs (hPDLCs. Materials and Methods. STRO-1 positive hPDLCs were sorted and the sorted cells were expanded and compared with their unsorted parental cells. Thereafter, hPDLCs were treated with or without Wnt3a and the cell proliferation, self-renewal, and osteogenic differentiation were evaluated. Results. No differences were measured between the expanded STRO-1-sorted cells and unsorted parental cells in terms of proliferation, CFU, and mineralization capacity. Wnt3a enhanced the proliferation and self-renewal ability of hPDLCs significantly as displayed by higher DNA content values, a shorter cell population doubling time, and higher expression of the self-renewal gene Oct4. Moreover, Wnt3a promoted the expansion of hPDLCs for 5 passages without affecting cell proliferation, CFU, and osteogenic capacity. Conclusions. Expanded STRO-1-sorted hPDLCs showed no superiority compared to their unsorted parental cells. On the other hand, Wnt3a promotes the efficient hPDLC expansion and retains the self-renewal and osteogenic differentiation capacity.

  2. Accuracy of the Fluorescence-Activated Cell Sorting Assay for the Aquaporin-4 Antibody (AQP4-Ab): Comparison with the Commercial AQP4-Ab Assay Kit

    Science.gov (United States)

    Kim, Yoo-Jin; Cheon, So Young; Kim, Boram; Jung, Kyeong Cheon; Park, Kyung Seok

    2016-01-01

    Background The aquaporin-4 antibody (AQP4-Ab) is a disease-specific autoantibody to neuromyelitis optica (NMO). We aimed to evaluate the accuracy of the FACS assay in detecting the AQP4-Ab compared with the commercial cell-based assay (C-CBA) kit. Methods Human embryonic kidney-293 cells were transfected with human aquaporin-4 (M23) cDNA. The optimal cut off values of FACS assay was tested using 1123 serum samples from patients with clinically definite NMO, those at high risk for NMO, patients with multiple sclerosis, patients with other idiopathic inflammatory demyelinating diseases, and negative controls. The accuracy of FACS assay and C-CBA were compared in consecutive 225 samples that were collected between January 2014 and June 2014. Results With a cut-off value of MFIi of 3.5 and MFIr of 2.0, the receiver operating characteristic curve for the FACS assay showed an area under the curve of 0.876. Among 225 consecutive sera, the FACS assay and C-CBA had a sensitivity of 77.3% and 69.7%, respectively, in differentiating the sera of definite NMO patients from sera of controls without IDD or of MS. Both assay had a good specificity of 100% in it. The overall positivity of the C-CBA among FACS-positive sera was 81.5%; moreover, its positivity was low as 50% among FACS-positive sera with relatively low MFIis. Conclusions Both the FACS assay and C-CBA are sensitive and highly specific assays in detecting AQP4-Ab. However, in some sera with relatively low antibody titer, FACS-assay can be a more sensitive assay option. In real practice, complementary use of FACS assay and C-CBA will benefit the diagnosis of NMO patients, because the former can be more sensitive among low titer sera and the latter are easier to use therefore can be widely used. PMID:27658059

  3. Parallel particle identification and separation for active optical sorting

    DEFF Research Database (Denmark)

    Perch-Nielsen, Ivan R.; Palima, Darwin; Dam, Jeppe Seidelin

    2009-01-01

    matched with a rapidly reconfigurable optical sorting field. We demonstrate the potential of such a system using colloidal polystyrene microspheres. By combining machine vision with a parallel add-on optical manipulation scheme, we were able to move identified particles over a distance of several hundred...

  4. Purification of Immune Cell Populations from Freshly Isolated Murine Tumors and Organs by Consecutive Magnetic Cell Sorting and Multi-parameter Flow Cytometry-Based Sorting.

    Science.gov (United States)

    Salvagno, Camilla; de Visser, Karin E

    2016-01-01

    It is well established that tumors evolve together with nonmalignant cells, such as fibroblasts, endothelial cells, and immune cells. These cells constantly entangle and interact with each other creating the tumor microenvironment. Immune cells can exert both tumor-promoting and tumor-protective functions. Detailed phenotypic and functional characterization of intra-tumoral immune cell subsets has become increasingly important in the field of cancer biology and cancer immunology. In this chapter, we describe a method for isolation of viable and pure immune cell subsets from freshly isolated murine solid tumors and organs. First, we describe a protocol for the generation of single-cell suspensions from tumors and organs using mechanical and enzymatic strategies. In addition, we describe how immune cell subsets can be purified by consecutive magnetic cell sorting and multi-parameter flow cytometry-based cell sorting.

  5. Postendocytic sorting of constitutively internalized dopamine transporter in cell lines and dopaminergic neurons

    DEFF Research Database (Denmark)

    Eriksen, Jacob; Bjørn-Yoshimoto, Walden Emil; Jørgensen, Trine Nygaard;

    2010-01-01

    The dopamine transporter (DAT) mediates reuptake of released dopamine and is the target for psychostimulants, such as cocaine and amphetamine. DAT undergoes marked constitutive endocytosis, but little is known about the fate and sorting of the endocytosed transporter. To study DAT sorting in cell...

  6. A kinetic mechanism for cell sorting based on local variations in cell motility

    Science.gov (United States)

    Strandkvist, Charlotte; Juul, Jeppe; Baum, Buzz; Kabla, Alexandre J.; Duke, Tom

    2014-01-01

    Our current understanding of cell sorting relies on physical difference, either in the interfacial properties or motile force, between cell types. But is such asymmetry a prerequisite for cell sorting? We test this using a minimal model in which the two cell populations are identical with respect to their physical properties and differences in motility arise solely from how cells interact with their surroundings. The model resembles the Schelling model used in social sciences to study segregation phenomena at the scale of societies. Our results demonstrate that segregation can emerge solely from cell motility being a dynamic property that changes in response to the local environment of the cell, but that additional mechanisms are necessary to reproduce the envelopment behaviour observed in vitro. The time course of segregation follows a power law, in agreement with the scaling reported from experiment and in other models of motility-driven segregation. PMID:25485079

  7. Multiparametric flow cytometry and cell sorting for the assessment of viable, injured, and dead bifidobacterium cells during bile salt stress.

    Science.gov (United States)

    Amor, Kaouther Ben; Breeuwer, Pieter; Verbaarschot, Patrick; Rombouts, Frank M; Akkermans, Antoon D L; De Vos, Willem M; Abee, Tjakko

    2002-11-01

    Using a flow cytometry-based approach, we assessed the viability of Bifidobacterium lactis DSM 10140 and Bifidobacterium adolescentis DSM 20083 during exposure to bile salt stress. Carboxyfluorescein diacetate (cFDA), propidium iodide (PI), and oxonol [DiBAC4(3)] were used to monitor esterase activity, membrane integrity, and membrane potential, respectively, as indicators of bacterial viability. Single staining with these probes rapidly and noticeably reflected the behavior of the two strains during stress exposure. However, the flow cytometry results tended to overestimate the viability of the two strains compared to plate counts, which appeared to be related to the nonculturability of a fraction of the population as a result of sublethal injury caused by bile salts. When the cells were simultaneously stained with cFDA and PI, flow cytometry and cell sorting revealed a striking physiological heterogeneity within the stressed bifidobacterium population. Three subpopulations could be identified based on their differential uptake of the probes: cF-stained, cF and PI double-stained, and PI-stained subpopulations, representing viable, injured, and dead cells, respectively. Following sorting and recovery, a significant fraction of the double-stained subpopulation (40%) could resume growth on agar plates. Our results show that in situ assessment of the physiological activity of stressed bifidobacteria using multiparameter flow cytometry and cell sorting may provide a powerful and sensitive tool for assessment of the viability and stability of probiotics.

  8. Chemokines as novel and versatile reagents for flow cytometry and cell sorting.

    Science.gov (United States)

    Le Brocq, Michelle L; Fraser, Alasdair R; Cotton, Graham; Woznica, Kerry; McCulloch, Clare V; Hewit, Kay D; McKimmie, Clive S; Nibbs, Robert J B; Campbell, John D M; Graham, Gerard J

    2014-06-15

    Cell therapy regimens are frequently compromised by low-efficiency cell homing to therapeutic niches. Improvements in this regard would enhance effectiveness of clinically applicable cell therapy. The major regulators of tissue-specific cellular migration are chemokines, and therefore selection of therapeutic cellular populations for appropriate chemokine receptor expression would enhance tissue-homing competence. A number of practical considerations preclude the use of Abs in this context, and alternative approaches are required. In this study, we demonstrate that appropriately labeled chemokines are at least as effective in detecting their cognate receptors as commercially available Abs. We also demonstrate the utility of biotinylated chemokines as cell-sorting reagents. Specifically, we demonstrate, in the context of CCR7 (essential for lymph node homing of leukocytes), the ability of biotinylated CCL19 with magnetic bead sorting to enrich for CCR7-expressing cells. The sorted cells demonstrate improved CCR7 responsiveness and lymph node-homing capability, and the sorting is effective for both T cells and dendritic cells. Importantly, the ability of chemokines to detect CCR7, and sort for CCR7 positivity, crosses species being effective on murine and human cells. This novel approach to cell sorting is therefore inexpensive, versatile, and applicable to numerous cell therapy contexts. We propose that this represents a significant technological advance with important therapeutic implications.

  9. Embryonic and induced pluripotent stem cell staining and sorting with the live-cell fluorescence imaging probe CDy1.

    Science.gov (United States)

    Kang, Nam-Young; Yun, Seong-Wook; Ha, Hyung-Ho; Park, Sung-Jin; Chang, Young-Tae

    2011-06-30

    Detecting and isolating specific types of cells is crucial to understanding a variety of biological processes, including development, aging, regeneration and pathogenesis; this understanding, in turn, allows the use of cells for therapeutic purposes, for which stem cells have emerged recently as invaluable materials. The current methods of isolation and characterization of stem cells depend on cell morphology in culture or on immunostaining of specific markers. These methods are, however, time consuming and involve the use of antibodies that may often make the cells unsuitable for further study. We recently developed a fluorescent small molecule named CDy1 (compound of designation yellow 1) that selectively stains live embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). This protocol describes detailed procedures for staining ESC and iPSC in live conditions and for fluorescence-activated cell sorting (FACS) of ESC using CDy1. Cell staining, image acquisition and FACS can be done within 6 h.

  10. Real-time fluorescence lifetime actuation for cell sorting using a CMOS SPAD silicon photomultiplier.

    Science.gov (United States)

    Rocca, Francescopaolo Mattioli Della; Nedbal, Jakub; Tyndall, David; Krstajić, Nikola; Li, David Day-Uei; Ameer-Beg, Simon M; Henderson, Robert K

    2016-02-15

    Time-correlated single photon counting (TCSPC) is a fundamental fluorescence lifetime measurement technique offering high signal to noise ratio (SNR). However, its requirement for complex software algorithms for histogram processing restricts throughput in flow cytometers and prevents on-the-fly sorting of cells. We present a single-point digital silicon photomultiplier (SiPM) detector accomplishing real-time fluorescence lifetime-activated actuation targeting cell sorting applications in flow cytometry. The sensor also achieves burst-integrated fluorescence lifetime (BIFL) detection by TCSPC. The SiPM is a single-chip complementary metal-oxide-semiconductor (CMOS) sensor employing a 32×32 single-photon avalanche diode (SPAD) array and eight pairs of time-interleaved time to digital converters (TI-TDCs) with a 50 ps minimum timing resolution. The sensor's pile-up resistant embedded center of mass method (CMM) processor accomplishes low-latency measurement and thresholding of fluorescence lifetime. A digital control signal is generated with a 16.6 μs latency for cell sorter actuation allowing a maximum cell throughput of 60,000 cells per second and an error rate of 0.6%.

  11. Draft Genome Sequence of Uncultivated Firmicutes (Peptococcaceae SCADC) Single Cells Sorted from Methanogenic Alkane-Degrading Cultures

    Science.gov (United States)

    Tan, BoonFei; Charchuk, Rhianna; Li, Carmen; Nesbø, Camilla; Abu Laban, Nidal

    2014-01-01

    The draft genome of an uncultivated bacterium affiliated with the Peptococcaceae was reconstructed by co-assembling Illumina MiSeq sequences from three single cells sorted by microfluidics from two methanogenic alkane-degrading cultures. Peptococcaceae SCADC (short-chain alkane-degrading culture) may be genetically capable of anaerobic alkane activation by fumarate addition in the absence of sulfate. PMID:25212628

  12. Investigating cell sorting and analysis of the proprietary cell-BOCS platform

    DEFF Research Database (Denmark)

    Carrissemoux, Caro; Beunis, Filip; Glückstad, Jesper

    2016-01-01

    This project comprises investigation of cell sorting, of both artificial beads and biological cells, and analysis of the proprietary cell-BOCS - Bio Optofluidic Cell Sorter, a table top cell sorter using optical manipulation. The analysis is more specific on broadening the ways of detection of ce...... modulated laser beam. The anticipated project output is a cheap, compact and easy-to-operate cell sorter that is able to detect and manipulate fluorescent microspheres, fluorescent labelled yeast cells and potentially erythrocytes in a microfluidic stream....

  13. High-throughput magnetic flow sorting of human cells selected on the basis of magnetophoretic mobility

    Science.gov (United States)

    Reece, Lisa M.; Sanders, Lehanna; Kennedy, David; Guernsey, Byron; Todd, Paul; Leary, James F.

    2010-02-01

    We have shown the potential of a new method for optimizing the separation of human stem cell subsets from peripheral blood based on a novel cell labeling technique that leverages the capabilities of a new commercially available high speed magnetic cell sorting system (IKOTECH LLC, New Albany, IN). This new system sorts cells in a continuously flowing manner using a Quadrupole Magnetic cell Sorter (QMS). The sorting mechanism is based upon the magnetophoretic mobility of the cells, a property related to the relative binding distributions of magnetic particles per cell, as determined by the utilization of a Magnetic Cell Tracking Velocimeter (MCTV). KG-1 cells were competitively labeled with anti-CD34 magnetic beads and anti-CD34 FITC to obtain an optimal level of magnetophoretic mobility as visualized by the MCTV for high throughput sort recovery in the QMS. In QMS sorting, the concept of split-flow thin channel (SPLITT) separation technology is applied by having a sample stream enter a vertical annular flow channel near the channel's interior wall followed by another sheath flow entering near the exterior wall. The two flows are initially separated by a flow splitter. They pass through the bore of a Halbach permanent quadrupole magnet assembly, which draws magnetized cells outward and deflects them into a positive outflow, while negative cells continue straight out via the inner flow lamina. QMS sorts cells based upon their magnetophoretic mobility, or the velocity of a cell per unit ponderomotive force, the counterpart of fluorescence intensity in flow cytometry. The magnetophoretic mobility distribution of a cell population, measured by automated MCTV, is used as input data for the algorithmic control of sample, sheath, and outlet flow velocities of the QMS. In this study, the relative binding distributions of magnetic particles per cell were determined by MCTV using novel sorting and sizing algorithms. The resulting mobility histograms were used to set the QMS

  14. Non-cyanobacterial nifH phylotypes in the North Pacific Subtropical Gyre detected by flow-cytometry cell sorting

    DEFF Research Database (Denmark)

    Bombar, Deniz; Turk-Kubo, Kendra A; Robidart, Julie

    2013-01-01

    In contrast to cyanobacteria, the significance of bacteria and archaea in oceanic N2 fixation remains unknown, apart from the knowledge that their nitrogenase (nifH) genes are diverse, present in all oceans and at least occasionally expressed. Non-cyanobacterial nifH sequences often occur...... as contamination from reagents and other sources, complicating the detection and interpretation of environmental phylotypes. We amplified and sequenced partial nifH gene fragments directly from cell populations sorted by fluorescence activated cell sorting from water collected in the North Pacific Subtropical Gyre...... (NPSG). Sequences recovered (195 total) included presumed heterotrophic or photoheterotrophic non-cyanobacterial nifH phylotypes previously unreported in the NPSG. A nifH sequence previously found in the South Pacific Gyre (HM210397) was exclusively recovered from sorted picoeukaryote populations...

  15. A cell sorting and trapping microfluidic device with an interdigital channel

    Directory of Open Access Journals (Sweden)

    Jing Tu

    2016-12-01

    Full Text Available The growing interest in cell sorting and trapping is driving the demand for high performance technologies. Using labeling techniques or external forces, cells can be identified by a series of methods. However, all of these methods require complicated systems with expensive devices. Based on inherent differences in cellular morphology, cells can be sorted by specific structures in microfluidic devices. The weir filter is a basic and efficient cell sorting and trapping structure. However, in some existing weir devices, because of cell deformability and high flow velocity in gaps, trapped cells may become stuck or even pass through the gaps. Here, we designed and fabricated a microfluidic device with interdigital channels for cell sorting and trapping. The chip consisted of a sheet of silicone elastomer polydimethylsiloxane and a sheet of glass. A square-wave-like weir was designed in the middle of the channel, comprising the interdigital channels. The square-wave pattern extended the weir length by three times with the channel width remaining constant. Compared with a straight weir, this structure exhibited a notably higher trapping capacity. Interdigital channels provided more space to slow down the rate of the pressure decrease, which prevented the cells from becoming stuck in the gaps. Sorting a mixture K562 and blood cells to trap cells demonstrated the efficiency of the chip with the interdigital channel to sort and trap large and less deformable cells. With stable and efficient cell sorting and trapping abilities, the chip with an interdigital channel may be widely applied in scientific research fields.

  16. Human Periodontal Ligament Derived Progenitor Cells: Effect of STRO-1 Cell Sorting and Wnt3a Treatment on Cell Behavior

    NARCIS (Netherlands)

    Yan, X.Z.; Both, S.K.; Yang, P.S.; Jansen, J.A.; Beucken, J.J.J.P van den; Yang, F.

    2014-01-01

    Objectives. STRO-1 positive periodontal ligament cells (PDLCs) and unsorted PDLCs have demonstrated potential for periodontal regeneration, but the comparison between unsorted cells and the expanded STRO-1 sorted cells has never been reported. Additionally, Wnt3a is involved in cell proliferation th

  17. Cell Sorting and Noise-Induced Cell Plasticity Coordinate to Sharpen Boundaries between Gene Expression Domains

    Science.gov (United States)

    2017-01-01

    A fundamental question in biology is how sharp boundaries of gene expression form precisely in spite of biological variation/noise. Numerous mechanisms position gene expression domains across fields of cells (e.g. morphogens), but how these domains are refined remains unclear. In some cases, domain boundaries sharpen through differential adhesion-mediated cell sorting. However, boundaries can also sharpen through cellular plasticity, with cell fate changes driven by up- or down-regulation of gene expression. In this context, we have argued that noise in gene expression can help cells transition to the correct fate. Here we investigate the efficacy of cell sorting, gene expression plasticity, and their combination in boundary sharpening using multi-scale, stochastic models. We focus on the formation of hindbrain segments (rhombomeres) in the developing zebrafish as an example, but the mechanisms investigated apply broadly to many tissues. Our results indicate that neither sorting nor plasticity is sufficient on its own to sharpen transition regions between different rhombomeres. Rather the two have complementary strengths and weaknesses, which synergize when combined to sharpen gene expression boundaries. PMID:28135279

  18. Cell detachment and label-free cell sorting using modulated surface acoustic waves (SAW) in droplet-based microfluidics

    CERN Document Server

    Bussonnière, Adrien; Baudoin, Michael; Bou-Matar, Olivier; Grandbois, Michel; Charette, Paul; Renaudin, Alan

    2014-01-01

    We present a droplet-based surface acoustic wave (SAW) system designed to viably detach biological cells from a surface and sort cell types based on differences in adhesion strength (adhesion contrast), without the need to label cells with molecular markers. The system uses modulated SAW to generate pulsatile flows in the droplets and efficiently detach the cells, thereby minimizing SAW excitation power and exposure time. As a proof-of-principle, the system is shown to efficiently sort HEK 293 from A7r5 cells based on adhesion contrast. Results are obtained in minutes with sorting purity and efficiency reaching 97 % and 95 %, respectively.

  19. IAP-Based Cell Sorting Results in Homogeneous Transplantable Dopaminergic Precursor Cells Derived from Human Pluripotent Stem Cells.

    Science.gov (United States)

    Lehnen, Daniela; Barral, Serena; Cardoso, Tiago; Grealish, Shane; Heuer, Andreas; Smiyakin, Andrej; Kirkeby, Agnete; Kollet, Jutta; Cremer, Harold; Parmar, Malin; Bosio, Andreas; Knöbel, Sebastian

    2017-09-19

    Human pluripotent stem cell (hPSC)-derived mesencephalic dopaminergic (mesDA) neurons can relieve motor deficits in animal models of Parkinson's disease (PD). Clinical translation of differentiation protocols requires standardization of production procedures, and surface-marker-based cell sorting is considered instrumental for reproducible generation of defined cell products. Here, we demonstrate that integrin-associated protein (IAP) is a cell surface marker suitable for enrichment of hPSC-derived mesDA progenitor cells. Immunomagnetically sorted IAP(+) mesDA progenitors showed increased expression of ventral midbrain floor plate markers, lacked expression of pluripotency markers, and differentiated into mature dopaminergic (DA) neurons in vitro. Intrastriatal transplantation of IAP(+) cells sorted at day 16 of differentiation in a rat model of PD resulted in functional recovery. Grafts from sorted IAP(+) mesDA progenitors were more homogeneous in size and DA neuron density. Thus, we suggest IAP-based sorting for reproducible prospective enrichment of mesDA progenitor cells in clinical cell replacement strategies. Copyright © 2017 Miltenyi Biotec GmbH. Published by Elsevier Inc. All rights reserved.

  20. Non-destructive on-chip cell sorting system with real-time microscopic image processing

    Directory of Open Access Journals (Sweden)

    Ichiki Takanori

    2004-06-01

    Full Text Available Abstract Studying cell functions for cellomics studies often requires the use of purified individual cells from mixtures of various kinds of cells. We have developed a new non-destructive on-chip cell sorting system for single cell based cultivation, by exploiting the advantage of microfluidics and electrostatic force. The system consists of the following two parts: a cell sorting chip made of poly-dimethylsiloxane (PDMS on a 0.2-mm-thick glass slide, and an image analysis system with a phase-contrast/fluorescence microscope. The unique features of our system include (i identification of a target from sample cells is achieved by comparison of the 0.2-μm-resolution phase-contrast and fluorescence images of cells in the microchannel every 1/30 s; (ii non-destructive sorting of target cells in a laminar flow by application of electrostatic repulsion force for removing unrequited cells from the one laminar flow to the other; (iii the use of agar gel for electrodes in order to minimize the effect on cells by electrochemical reactions of electrodes, and (iv pre-filter, which was fabricated within the channel for removal of dust contained in a sample solution from tissue extracts. The sorting chip is capable of continuous operation and we have purified more than ten thousand cells for cultivation without damaging them. Our design has proved to be very efficient and suitable for the routine use in cell purification experiments.

  1. The viral spike protein is not involved in the polarized sorting of coronaviruses in epithelial cells

    NARCIS (Netherlands)

    Rossen, J W; de Beer, R; Godeke, G J; Raamsman, M J; Horzinek, M C; Vennema, H; Rottier, P J

    1998-01-01

    Coronaviruses are assembled by budding into a pre-Golgi compartment from which they are transported along the secretory pathway to leave the cell. In cultured epithelial cells, they are released in a polarized fashion; depending on the virus and cell type, they are sorted preferentially either to th

  2. Multi-parameter flow cytometry and cell sorting reveal extensive physiological heterogeneity in Bacillus cereus batch cultures.

    Science.gov (United States)

    Want, Andrew; Hancocks, Helen; Thomas, Colin R; Stocks, Stuart M; Nebe-von-Caron, Gerhard; Hewitt, Christopher J

    2011-07-01

    Based on two staining protocols, DiOC(6)(3)/propidium iodide (PI) and RedoxSensor Green (an indicator of bacterial reductase activity)/PI, multi-parameter flow cytometry and cell sorting has identified at least four distinguishable physiological states during batch cultures of Bacillus cereus. Furthermore, dependent on the position in the growth curve, single cells gave rise to varying numbers of colonies when sorted individually onto nutrient agar plates. These growing colonies derived from a single cell had widely different lag phases, inferred from differences in colony size. This further highlights the complex population dynamics of bacterial monocultures and further demonstrates that individual bacterial cells in a culture respond in markedly dissimilar ways to the environment, resulting in a physiologically heterogenous and dynamic population.

  3. Mean-cluster approach indicates cell sorting time scales are determined by collective dynamics

    Science.gov (United States)

    Beatrici, Carine P.; de Almeida, Rita M. C.; Brunnet, Leonardo G.

    2017-03-01

    Cell migration is essential to cell segregation, playing a central role in tissue formation, wound healing, and tumor evolution. Considering random mixtures of two cell types, it is still not clear which cell characteristics define clustering time scales. The mass of diffusing clusters merging with one another is expected to grow as td /d +2 when the diffusion constant scales with the inverse of the cluster mass. Cell segregation experiments deviate from that behavior. Explanations for that could arise from specific microscopic mechanisms or from collective effects, typical of active matter. Here we consider a power law connecting diffusion constant and cluster mass to propose an analytic approach to model cell segregation where we explicitly take into account finite-size corrections. The results are compared with active matter model simulations and experiments available in the literature. To investigate the role played by different mechanisms we considered different hypotheses describing cell-cell interaction: differential adhesion hypothesis and different velocities hypothesis. We find that the simulations yield normal diffusion for long time intervals. Analytic and simulation results show that (i) cluster evolution clearly tends to a scaling regime, disrupted only at finite-size limits; (ii) cluster diffusion is greatly enhanced by cell collective behavior, such that for high enough tendency to follow the neighbors, cluster diffusion may become independent of cluster size; (iii) the scaling exponent for cluster growth depends only on the mass-diffusion relation, not on the detailed local segregation mechanism. These results apply for active matter systems in general and, in particular, the mechanisms found underlying the increase in cell sorting speed certainly have deep implications in biological evolution as a selection mechanism.

  4. Computational modeling reveals that a combination of chemotaxis and differential adhesion leads to robust cell sorting during tissue patterning.

    Directory of Open Access Journals (Sweden)

    Rui Zhen Tan

    Full Text Available Robust tissue patterning is crucial to many processes during development. The "French Flag" model of patterning, whereby naïve cells in a gradient of diffusible morphogen signal adopt different fates due to exposure to different amounts of morphogen concentration, has been the most widely proposed model for tissue patterning. However, recently, using time-lapse experiments, cell sorting has been found to be an alternative model for tissue patterning in the zebrafish neural tube. But it remains unclear what the sorting mechanism is. In this article, we used computational modeling to show that two mechanisms, chemotaxis and differential adhesion, are needed for robust cell sorting. We assessed the performance of each of the two mechanisms by quantifying the fraction of correct sorting, the fraction of stable clusters formed after correct sorting, the time needed to achieve correct sorting, and the size variations of the cells having different fates. We found that chemotaxis and differential adhesion confer different advantages to the sorting process. Chemotaxis leads to high fraction of correct sorting as individual cells will either migrate towards or away from the source depending on its cell type. However after the cells have sorted correctly, there is no interaction among cells of the same type to stabilize the sorted boundaries, leading to cell clusters that are unstable. On the other hand, differential adhesion results in low fraction of correct clusters that are more stable. In the absence of morphogen gradient noise, a combination of both chemotaxis and differential adhesion yields cell sorting that is both accurate and robust. However, in the presence of gradient noise, the simple combination of chemotaxis and differential adhesion is insufficient for cell sorting; instead, chemotaxis coupled with delayed differential adhesion is required to yield optimal sorting.

  5. Multifunctional assembly of micrometer-sized colloids for cell sorting.

    Science.gov (United States)

    Nie, Chenyao; Wang, Bing; Zhang, Jiangyan; Cheng, Yongqiang; Lv, Fengting; Liu, Libing; Wang, Shu

    2015-06-03

    Compared to the extensively studied nanometer-sized colloids, less attention has been paid to the assembly of micrometer-sized colloids with multifunctional characteristics. To address this need, a bottom-up approach is developed for constructing self-assemblies of micrometer-sized magnetic colloids possessing multifunctionality, including magnetic, optical, and biological activities. Biotinylated oligo (p-phenylene vinylene) (OPV) derivatives are designed to mediate the self-assembly of streptavidin-modified magnetic beads. The optical element OPV derivatives provide a fluorescence imaging ability for tracing the assembly process. Target cells can be recognized and assembled by the colloidal assembly with bioactive element antibodies. The colloidal assembly reveals better cell isolation performance by its amplified magnetic response in comparison to monodisperse colloids. The self-assembly of micrometer-sized magnetic colloids through a combination of different functional ingredients to realize multifunction is conceptually simple and easy to achieve. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Accurate determination of plasmid copy number of flow-sorted cells using droplet digital PCR.

    Science.gov (United States)

    Jahn, Michael; Vorpahl, Carsten; Türkowsky, Dominique; Lindmeyer, Martin; Bühler, Bruno; Harms, Hauke; Müller, Susann

    2014-06-17

    Many biotechnological processes rely on the expression of a plasmid-based target gene. A constant and sufficient number of plasmids per cell is desired for efficient protein production. To date, only a few methods for the determination of plasmid copy number (PCN) are available, and most of them average the PCN of total populations disregarding heterogeneous distributions. Here, we utilize the highly precise quantification of DNA molecules by droplet digital PCR (ddPCR) and combine it with cell sorting using flow cytometry. A duplex PCR assay was set up requiring only 1000 sorted cells for precise determination of PCN. The robustness of this method was proven by thorough optimization of cell sorting, cell disruption, and PCR conditions. When non plasmid-harboring cells of Pseudomonas putida KT2440 were spiked with different dilutions of the expression plasmid pA-EGFP_B, a PCN from 1 to 64 could be accurately detected. As a proof of principle, induced cultures of P. putida KT2440 producing an EGFP-fused model protein by means of the plasmid pA-EGFP_B were investigated by flow cytometry and showed two distinct subpopulations, fluorescent and nonfluorescent cells. These two subpopulations were sorted for PCN determination with ddPCR. A remarkably diverging plasmid distribution was found within the population, with nonfluorescent cells showing a much lower PCN (≤1) than fluorescent cells (PCN of up to 5) under standard conditions.

  7. Harnessing single cell sorting to identify cell division genes and regulators in bacteria.

    Directory of Open Access Journals (Sweden)

    Catherine Burke

    Full Text Available Cell division is an essential cellular process that requires an array of known and unknown proteins for its spatial and temporal regulation. Here we develop a novel, high-throughput screening method for the identification of bacterial cell division genes and regulators. The method combines the over-expression of a shotgun genomic expression library to perturb the cell division process with high-throughput flow cytometry sorting to screen many thousands of clones. Using this approach, we recovered clones with a filamentous morphology for the model bacterium, Escherichia coli. Genetic analysis revealed that our screen identified both known cell division genes, and genes that have not previously been identified to be involved in cell division. This novel screening strategy is applicable to a wide range of organisms, including pathogenic bacteria, where cell division genes and regulators are attractive drug targets for antibiotic development.

  8. A method for high purity sorting of rare cell subsets applied to TDC

    OpenAIRE

    2013-01-01

    TDC are a recently described subset of polyclonal αβ T-cells with dendritic cell properties. Because of their low number in peripheral immune compartments, isolation and characterization of TDC with existing purification methods is technically challenging. Here we describe a customized gating strategy and a flow cytometry-based cell sorting protocol for isolation of TDC. The protocol was developed because, despite very conservative gating for dead-cell and doublet exclusion, cells obtained wi...

  9. Identification, visualization, and sorting of translationally active microbial consortia from deep-sea methane seeps

    Science.gov (United States)

    Hatzenpichler, R.; Connon, S. A.; Goudeau, D.; Malmstrom, R.; Woyke, T.; Orphan, V. J.

    2015-12-01

    Within the past few years, great progress has been made in tapping the genomes of individual cells separated from environmental samples. Unfortunately, however, most often these efforts have been target blind, as they did not pre-select for taxa of interest or focus on metabolically active cells that could be considered key species of the system at the time. This problem is particularly pronounced in low-turnover systems such as deep sea sediments. In an effort to tap the genetic potential hidden within functionally active cells, we have recently developed an approach for the in situ fluorescent tracking of protein synthesis in uncultured cells via bioorthogonal non-canonical amino acid-tagging (BONCAT). This technique depends on the incorporation of synthetic amino acids that carry chemically modifiable tags into newly made proteins, which later can be visualized via click chemistry-mediated fluorescence-labeling. BONCAT is thus able to specifically target proteins that have been expressed in reaction to an experimental condition. We are particularly interested in using BONCAT to understand the functional potential of slow-growing syntrophic consortia of anaerobic methanotrophic archaea and sulfate-reducing bacteria which together catalyze the anaerobic oxidation of methane (AOM) in marine methane seeps. In order to specifically target consortia that are active under varying environmental regimes, we are studying different subpopulations of these inter-domain consortia via a combination of BONCAT with rRNA-targeted FISH. We then couple the BONCAT-enabled staining of active consortia with their separation from inactive members of the community via fluorescence-activated cell-sorting (FACS) and metagenomic sequencing of individual consortia. Using this approach, we were able to identify previously unrecognized AOM-partnerships. By comparing the mini-metagenomes obtained from individual consortia with each other we are starting to gain a more hollistic understanding

  10. Using pico-LCoS SLMs for high speed cell sorting

    DEFF Research Database (Denmark)

    Bañas, Andrew Rafael; Aabo, Thomas; Palima, Darwin

    2012-01-01

    We propose the use of consumer pico projectors as cost effective spatial light modulators in cell sorting applications. The matched filtering Generalized Phase Contrast (mGPC) beam shaping method is used to produce high intensity optical spots for trapping and catapulting cells. A pico projector...

  11. HLA-targeted flow cytometric sorting of blood cells allows separation of pure and viable microchimeric cell populations.

    Science.gov (United States)

    Drabbels, Jos J M; van de Keur, Carin; Kemps, Berit M; Mulder, Arend; Scherjon, Sicco A; Claas, Frans H J; Eikmans, Michael

    2011-11-10

    Microchimerism is defined by the presence of low levels of nonhost cells in a person. We developed a reliable method for separating viable microchimeric cells from the host environment. For flow cytometric cell sorting, HLA antigens were targeted with human monoclonal HLA antibodies (mAbs). Optimal separation of microchimeric cells (present at a proportion as low as 0.01% in artificial mixtures) was obtained with 2 different HLA mAbs, one targeting the chimeric cells and the other the background cells. To verify purity of separated cell populations, flow-sorted fractions of 1000 cells were processed for DNA analysis by HLA-allele-specific and Y-chromosome-directed real-time quantitative PCR assays. After sorting, PCR signals of chimeric DNA markers in the positive fractions were significantly enhanced compared with those in the presort samples, and they were similar to those in 100% chimeric control samples. Next, we demonstrate applicability of HLA-targeted FACS sorting after pregnancy by separating chimeric maternal cells from child umbilical cord mononuclear cells. Targeting allelic differences with anti-HLA mAbs with FACS sorting allows maximal enrichment of viable microchimeric cells from a background cell population. The current methodology enables reliable microchimeric cell detection and separation in clinical specimens.

  12. Participation in Occupational Performance: Reliability and Validity of the Activity Card Sort.

    Science.gov (United States)

    Katz, Noomi; Karpin, Hanah; Lak, Arit; Furman, Tania; Hartman-Maeir, Adina

    2003-01-01

    A study assessed the reliability and validity of the Activity Card Sort (ACS) within different adult groups (n=263): healthy adults, healthy older adults, Alzheimer's caregivers, multiple sclerosis patients, and stroke survivors. Found that the ACS had high internal consistency for daily living and social-cultural activities and a lower…

  13. Acid tolerance of Streptococcus macedonicus as assessed by flow cytometry and single-cell sorting.

    Science.gov (United States)

    Papadimitriou, Konstantinos; Pratsinis, Harris; Nebe-von-Caron, Gerhard; Kletsas, Dimitris; Tsakalidou, Effie

    2007-01-01

    An in situ flow cytometric viability assay employing carboxyfluorescein diacetate and propidium iodide was used to identify Streptococcus macedonicus acid tolerance phenotypes. The logarithmic-phase acid tolerance response (L-ATR) was evident when cells were (i) left to autoacidify unbuffered medium, (ii) transiently exposed to nonlethal acidic pH, or (iii) systematically grown under suboptimal acidic conditions (acid habituation). Stationary-phase ATR was also detected; this phenotype was gradually degenerated while cells resided at this phase. Single-cell analysis of S. macedonicus during induction of L-ATR revealed heterogeneity in both the ability and the rate of tolerance acquisition within clonal populations. L-ATR was found to be partially dependent on de novo protein synthesis and compositional changes of the cell envelope. Interestingly, acid-habituated cells were interlaced in lengthier chains and exhibited an irregular pattern of active peptidoglycan biosynthesis sites when probed with BODIPY FL vancomycin. L-ATR caused cells to retain their membrane potential after lethal challenge, as judged by ratiometric analysis with oxonol [DiBAC(4)(3)]. Furthermore, F-ATPase was important during the induction of L-ATR, but in the case of a fully launched response, inhibition of F-ATPase affected acid resistance only partially. Activities of both F-ATPase and the glucose-specific phosphoenolpyruvate-dependent phosphotransferase system were increased after L-ATR induction, distinguishing S. macedonicus from oral streptococci. Finally, the in situ viability assessment was compared to medium-based recovery after single-cell sorting, revealing that the culturability of subpopulations with identical fluorescence characteristics is dependent on the treatments imposed to the cells prior to acid challenge.

  14. HURP permits MTOC sorting for robust meiotic spindle bipolarity, similar to extra centrosome clustering in cancer cells.

    Science.gov (United States)

    Breuer, Manuel; Kolano, Agnieszka; Kwon, Mijung; Li, Chao-Chin; Tsai, Ting-Fen; Pellman, David; Brunet, Stéphane; Verlhac, Marie-Hélène

    2010-12-27

    In contrast to somatic cells, formation of acentriolar meiotic spindles relies on the organization of microtubules (MTs) and MT-organizing centers (MTOCs) into a stable bipolar structure. The underlying mechanisms are still unknown. We show that this process is impaired in hepatoma up-regulated protein (Hurp) knockout mice, which are viable but female sterile, showing defective oocyte divisions. HURP accumulates on interpolar MTs in the vicinity of chromosomes via Kinesin-5 activity. By promoting MT stability in the spindle central domain, HURP allows efficient MTOC sorting into distinct poles, providing bipolarity establishment and maintenance. Our results support a new model for meiotic spindle assembly in which HURP ensures assembly of a central MT array, which serves as a scaffold for the genesis of a robust bipolar structure supporting efficient chromosome congression. Furthermore, HURP is also required for the clustering of extra centrosomes before division, arguing for a shared molecular requirement of MTOC sorting in mammalian meiosis and cancer cell division.

  15. Proliferation of sorted human and rat beta cells

    DEFF Research Database (Denmark)

    Parnaud, G; Bosco, D; Berney, T;

    2008-01-01

    The aim of the study was to determine whether purified beta cells can replicate in vitro and whether this is enhanced by extracellular matrix (ECM) and growth factors.......The aim of the study was to determine whether purified beta cells can replicate in vitro and whether this is enhanced by extracellular matrix (ECM) and growth factors....

  16. Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases

    Directory of Open Access Journals (Sweden)

    Arildo Nerys-Junior

    2014-01-01

    Full Text Available Engineered nucleases such as zinc finger nucleases (ZFN and transcription activator-like effector nucleases (TALEN are one of the most promising tools for modifying genomes. These site-specific enzymes cause double- strand breaks that allow gene disruption or gene insertion, thereby facilitating genetic manipulation. The major problem associated with this approach is the labor-intensive procedures required to screen and confirm the cellular modification by nucleases. In this work, we produced a TALEN that targets the human CCR5 gene and developed a heteroduplex mobility assay for HEK 293T cells to select positive colonies for sequencing. This approach provides a useful tool for the quick detection and easy assessment of nuclease activity.

  17. Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases.

    Science.gov (United States)

    Nerys-Junior, Arildo; Costa, Lendel C; Braga-Dias, Luciene P; Oliveira, Márcia; Rossi, Atila D; da Cunha, Rodrigo Delvecchio; Gonçalves, Gabriel S; Tanuri, Amilcar

    2014-03-01

    Engineered nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) are one of the most promising tools for modifying genomes. These site-specific enzymes cause double-strand breaks that allow gene disruption or gene insertion, thereby facilitating genetic manipulation. The major problem associated with this approach is the labor-intensive procedures required to screen and confirm the cellular modification by nucleases. In this work, we produced a TALEN that targets the human CCR5 gene and developed a heteroduplex mobility assay for HEK 293T cells to select positive colonies for sequencing. This approach provides a useful tool for the quick detection and easy assessment of nuclease activity.

  18. Overview of the New Flow Cytometry RG and Proposed Cell Sorting (FACS) Microarray study

    OpenAIRE

    DeLay, Monica; Lopez, Peter; Tighe, Scott

    2013-01-01

    The Flow Cytometry Research Group (FCRG) is the latest addition to the ABRF RG family. The RG is currently in its first year and has 9 members; many of whom are flow cytometrists new to the ABRF. The initial goal of the FCRG is to describe a method for the evaluation of cell stress or other deleterious perturbations caused by cell sorting across a wide range of cell types.

  19. High-throughput cell analysis and sorting technologies for clinical diagnostics and therapeutics

    Science.gov (United States)

    Leary, James F.; Reece, Lisa M.; Szaniszlo, Peter; Prow, Tarl W.; Wang, Nan

    2001-05-01

    A number of theoretical and practical limits of high-speed flow cytometry/cell sorting are important for clinical diagnostics and therapeutics. Three applications include: (1) stem cell isolation with tumor purging for minimal residual disease monitoring and treatment, (2) identification and isolation of human fetal cells from maternal blood for prenatal diagnostics and in-vitro therapeutics, and (3) high-speed library screening for recombinant vaccine production against unknown pathogens.

  20. Overview of the New Flow Cytometry RG and Proposed Cell Sorting (FACS) Microarray study

    OpenAIRE

    2013-01-01

    The Flow Cytometry Research Group (FCRG) is the latest addition to the ABRF RG family. The RG is currently in its first year and has 9 members; many of whom are flow cytometrists new to the ABRF. The initial goal of the FCRG is to describe a method for the evaluation of cell stress or other deleterious perturbations caused by cell sorting across a wide range of cell types.

  1. Raman tweezers in microfluidic systems for analysis and sorting of living cells

    Science.gov (United States)

    Pilát, Zdenëk; Ježek, Jan; Kaňka, Jan; Zemánek, Pavel

    2014-03-01

    We have devised an analytical and sorting system combining optical trapping with Raman spectroscopy in microfluidic environment in order to identify and sort biological objects, such as living cells of various prokaryotic and eukaryotic organisms. Our main objective was to create a robust and universal platform for non-contact sorting of microobjects based on their Raman spectral properties. This approach allowed us to collect information about the chemical composition of the objects, such as the presence and composition of lipids, proteins, or nucleic acids without using artificial chemical probes such as fluorescent markers. The non-destructive and non-contact nature of this optical analysis and manipulation allowed us to separate individual living cells of our interest in a sterile environment and provided the possibility to cultivate the selected cells for further experiments. We used differently treated cells of algae to test and demonstrate the function of our analytical and sorting system. The devised system could find its use in many medical, biotechnological, and biological applications.

  2. Sorting and biological characteristics analysis for side population cells in human primary hepatocellular carcinoma

    Science.gov (United States)

    Jiang, Yegui; Gao, Hucheng; Liu, Mingdong; Mao, Qing

    2016-01-01

    Hepatocellular carcinoma (HCC) is the fifth most common cause of the tumor worldwide, its incidence is increasing year by year. This study aims to investigate the sorting and biological characteristics of side population (SP) cells. Human HCC tissues used were obtained from patients undergoing surgical resection. SP cells were sorted using flow cytometry. Cell cycle assay, apoptosis assay and colony formation assay were performed to detect cell proliferation and apoptosis. Invasion assay was employed to examine SP cell invasion. Tumorigenicity assay was used to evaluate tumorigenicity. HCC related microRNAs (miRNA) were analyzed using Micro-array analysis. Target genes were predicted using miRNA database. GO analsis was employed to predict target gene function. Apoptosis percentage was lower and cell viability was higher in SP cells than non-SP (NSP) cells. Colony forming ability of SP cells was significantly higher than NSP cells. Transwell assay positive cells in SP cells were higher significantly than NSP cells. Tumorigenicity of SP cells was higher significantly than NSP cells. 107 differentially expression miRNA were discovered, including 45 up-expressed miRNAs and 62 down-expressed miRNAs in SP cells. Up-regulated hsa-miR-193b-3p and hsa-miR-505-3p predict 25 and 35 target genes, and correlated with 4 and 42 GO terms, respectively. Down-regulated hsa-miR-200a-3p, hsa-miR-194-5p, hsa-miR-130b-3p predict 133, 48 and 127 target genes, and correlate with 10, 7 and 109 GO terms, respectively. In conclusion, proliferation, colony formation, anti-apoptosis, self-renewal capavility, invasive characteristic and tumorigenicity in SP cells isolated from HCC tissues was higher compared to NSP cells. Therefore, sorted SP cells could characterize with biological functions of cancer stem cells.

  3. Application of sorting peripheral blood cells by flow cytometry in the detection of peripheral Mood cell sorting%流式细胞术分选外周血细胞方法的研究

    Institute of Scientific and Technical Information of China (English)

    郑美婧; 段静静; 苏丽萍; 王艳峰; 苏文

    2010-01-01

    Objective To apply fluo-rescencc-activated cell sorting(FACS) in sorting T lymphocyte (CD_3~+) and granulocyte (CD_(15)~+), which establish the separating method of series of cells from human peripheral blood, so that the scientific research and clinical research could be carried out were specifically. Methods 10 cases of normal peripheral blood were collected and T lymphocyte (CD_3~+) and granulocyte (CD_(15)~+) were stained with florescence conjugated antibodies. The positive cells were sorted by FACS. Results Before sorting the peripheral blood, the proportion of the T lymphocyte(CD_3~+) and granulocyte (CD_(15)~+) in the leukocyte is 48.8 % and 30.8 %; after sorting by FACS, the purity of T lymphocytes (CD_3~+) is up to 98 % and the recovery is about 95 %; the purity of granulocyte (CD_(15)~+) is up to 97 % and the recovery is about 96 %. Conclusion The FACS could Mlow us to quickly sort T lymphocyte (CD_3~+) and granulocyte (CD_(15)~+) with higher recovery and higher purity from the peripheral blood.%目的 应用荧光激活细胞分选技术(FACS)分选T淋巴细胞(CD_3~+)和粒细胞(CD_(25)~+),建立人类外周血各细胞亚群的分离方法,为基于各细胞亚群的科研和临床研究奠定实验基础.方法 收集10例健康人的外周血,利用抗体分别标定粒细胞及T淋巴细胞,应用FACS进一步分选出CD_3~+T淋巴细胞及CD_(15)~+粒细胞.结果 分选前外周血中CD_3~+T淋巴细胞约占白细胞总数的30.8%,CD_(15)~+粒细胞约占白细胞总数的48.8%;FACS分离纯化后的CD_3~+T淋巴细胞的纯度可达98%,回收率为95%;CD_(15)~+粒细胞的纯度可达97%,回收率约为96%.结论 应用FACS可从外周血中快速分离高纯度的CD_3~+T淋巴细胞及CD_(15)~+粒细胞.

  4. Computational cell model based on autonomous cell movement regulated by cell-cell signalling successfully recapitulates the "inside and outside" pattern of cell sorting

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    Ajioka Itsuki

    2007-09-01

    Full Text Available Abstract Background Development of multicellular organisms proceeds from a single fertilized egg as the combined effect of countless numbers of cellular interactions among highly dynamic cells. Since at least a reminiscent pattern of morphogenesis can be recapitulated in a reproducible manner in reaggregation cultures of dissociated embryonic cells, which is known as cell sorting, the cells themselves must possess some autonomous cell behaviors that assure specific and reproducible self-organization. Understanding of this self-organized dynamics of heterogeneous cell population seems to require some novel approaches so that the approaches bridge a gap between molecular events and morphogenesis in developmental and cell biology. A conceptual cell model in a computer may answer that purpose. We constructed a dynamical cell model based on autonomous cell behaviors, including cell shape, growth, division, adhesion, transformation, and motility as well as cell-cell signaling. The model gives some insights about what cellular behaviors make an appropriate global pattern of the cell population. Results We applied the model to "inside and outside" pattern of cell-sorting, in which two different embryonic cell types within a randomly mixed aggregate are sorted so that one cell type tends to gather in the central region of the aggregate and the other cell type surrounds the first cell type. Our model can modify the above cell behaviors by varying parameters related to them. We explored various parameter sets with which the "inside and outside" pattern could be achieved. The simulation results suggested that direction of cell movement responding to its neighborhood and the cell's mobility are important for this specific rearrangement. Conclusion We constructed an in silico cell model that mimics autonomous cell behaviors and applied it to cell sorting, which is a simple and appropriate phenomenon exhibiting self-organization of cell population. The model

  5. Specific rates of leucine incorporation by marine bacterioplantkon in the open Mediterranean Sea in summer using cell sorting

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    Talarmin, A.; van Wambeke, F.; Catala, P.; Courties, C.; Lebaron, P.

    2010-08-01

    Cell-specific leucine incorporation rates were determined in early summer across the open stratified Mediterranean Sea along vertical profiles from 0 to 200 m. During the period of our study, the bulk leucine incorporation rate was on average 5.0 ± 4.0 (n=31) pmol leu l-1 h-1. After 3H-radiolabeled leucine incorporation and SyBR Green I staining, populations were sorted using flow cytometry. Heterotrophic prokaryotes (Hprok) were divided in several clusters according to the cytometric properties of side scatter and green fluorescence of the cells: the low nucleic acid content cells (LNA) and the high nucleic acid content cells (HNA), with high size and low size (HNA-hs and HNA-ls, respectively). LNA cells represented 45 to 63% of the Hprok abundance between surface and 200 m, and significantly contributed to the bulk activity, from 17 to 55% all along the transect. The HNA/LNA ratio of cell-specific activities was on average 2.1 ± 0.7 (n=31). Among Hprok populations from surface samples (0 down to the deep chlorophyll depth, DCM), HNA-hs was mostly responsible for the leucine incorporation activity. Its cell-specific activity was up to 13.3 and 6.9-fold higher than that of HNA-ls and LNA, respectively, and it varied within a wide range of values (0.9-54.3×10-21 mol leu cell-1 h-1). At the opposite, ratios between the specific activities of the 3 populations tended to get closer to each other, below the DCM, implying a potentially higher homogeneity in activity of Hprok in the vicinity of nutriclines. Prochlorococcus cells were easily sorted near the DCM and displayed cell-specific activities equally high, sometimes higher than the HNA-hs group (2.5-55×10-21 mol leu cell-1 h-1). We then showed that all the sorted populations were key-players in leucine incorporation into proteins. The mixotrophic feature of certain photosynthetic prokaryotes and the non-negligible activity of LNA cells all over Mediterranean were reinforced.

  6. Specific rates of leucine incorporation by marine bacterioplantkon in the open Mediterranean Sea in summer using cell sorting

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    A. Talarmin

    2010-08-01

    Full Text Available Cell-specific leucine incorporation rates were determined in early summer across the open stratified Mediterranean Sea along vertical profiles from 0 to 200 m. During the period of our study, the bulk leucine incorporation rate was on average 5.0 ± 4.0 (n=31 pmol leu l−1 h−1. After 3H-radiolabeled leucine incorporation and SyBR Green I staining, populations were sorted using flow cytometry. Heterotrophic prokaryotes (Hprok were divided in several clusters according to the cytometric properties of side scatter and green fluorescence of the cells: the low nucleic acid content cells (LNA and the high nucleic acid content cells (HNA, with high size and low size (HNA-hs and HNA-ls, respectively. LNA cells represented 45 to 63% of the Hprok abundance between surface and 200 m, and significantly contributed to the bulk activity, from 17 to 55% all along the transect. The HNA/LNA ratio of cell-specific activities was on average 2.1 ± 0.7 (n=31. Among Hprok populations from surface samples (0 down to the deep chlorophyll depth, DCM, HNA-hs was mostly responsible for the leucine incorporation activity. Its cell-specific activity was up to 13.3 and 6.9-fold higher than that of HNA-ls and LNA, respectively, and it varied within a wide range of values (0.9–54.3×10−21 mol leu cell−1 h−1. At the opposite, ratios between the specific activities of the 3 populations tended to get closer to each other, below the DCM, implying a potentially higher homogeneity in activity of Hprok in the vicinity of nutriclines. Prochlorococcus cells were easily sorted near the DCM and displayed cell-specific activities equally high, sometimes higher than the HNA-hs group (2.5–55×10−21 mol leu cell−1 h−1. We then showed that all the sorted populations were key-players in leucine incorporation into proteins. The mixotrophic feature of

  7. [Chimerism analysis after allogeneic haematopoietic stem cell transplantation. Interest of cell sorting: general review].

    Science.gov (United States)

    Mollet, I; Giannoli, C; Rigal, D; Dubois, V

    2012-04-01

    Haematopoietic stem cells transplantation, widely used these last decades, represent the ultimate treatment resource for patients with haematological malignancies. Long range success of this treatment is particularly affected by relapse of the initial disease, graft rejection or graft versus host disease. Chimerism analysis after transplantation had been used since several years to document engraftment, to determine the risk of relapse and to adapt therapy promptly when necessary. Usefulness of this analysis for the outcome of transplanted patients, as well as the impact of using high sensitive techniques coupled with specific cell populations sorted have been demonstrated by retrospective studies. Follow-up of chimerism would allow to operate efficiently before the onset of clinical signs in leukaemic patients with high risk of relapse and to control the expression of minimal residual disease when specific molecular markers could not be monitored. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

  8. Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters.

    Science.gov (United States)

    Vorobjev, Ivan A; Buchholz, Kathrin; Prabhat, Prashant; Ketman, Kenneth; Egan, Elizabeth S; Marti, Matthias; Duraisingh, Manoj T; Barteneva, Natasha S

    2012-09-05

    Malaria remains a major cause of morbidity and mortality worldwide. Flow cytometry-based assays that take advantage of fluorescent protein (FP)-expressing malaria parasites have proven to be valuable tools for quantification and sorting of specific subpopulations of parasite-infected red blood cells. However, identification of rare subpopulations of parasites using green fluorescent protein (GFP) labelling is complicated by autofluorescence (AF) of red blood cells and low signal from transgenic parasites. It has been suggested that cell sorting yield could be improved by using filters that precisely match the emission spectrum of GFP. Detection of transgenic Plasmodium falciparum parasites expressing either tdTomato or GFP was performed using a flow cytometer with interchangeable optical filters. Parasitaemia was evaluated using different optical filters and, after optimization of optics, the GFP-expressing parasites were sorted and analysed by microscopy after cytospin preparation and by imaging cytometry. A new approach to evaluate filter performance in flow cytometry using two-dimensional dot blot was developed. By selecting optical filters with narrow bandpass (BP) and maximum position of filter emission close to GFP maximum emission in the FL1 channel (510/20, 512/20 and 517/20; dichroics 502LP and 466LP), AF was markedly decreased and signal-background improve dramatically. Sorting of GFP-expressing parasite populations in infected red blood cells at 90 or 95% purity with these filters resulted in 50-150% increased yield when compared to the standard filter set-up. The purity of the sorted population was confirmed using imaging cytometry and microscopy of cytospin preparations of sorted red blood cells infected with transgenic malaria parasites. Filter optimization is particularly important for applications where the FP signal and percentage of positive events are relatively low, such as analysis of parasite-infected samples with in the intention of gene

  9. Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters

    Directory of Open Access Journals (Sweden)

    Vorobjev Ivan A

    2012-09-01

    Full Text Available Abstract Background Malaria remains a major cause of morbidity and mortality worldwide. Flow cytometry-based assays that take advantage of fluorescent protein (FP-expressing malaria parasites have proven to be valuable tools for quantification and sorting of specific subpopulations of parasite-infected red blood cells. However, identification of rare subpopulations of parasites using green fluorescent protein (GFP labelling is complicated by autofluorescence (AF of red blood cells and low signal from transgenic parasites. It has been suggested that cell sorting yield could be improved by using filters that precisely match the emission spectrum of GFP. Methods Detection of transgenic Plasmodium falciparum parasites expressing either tdTomato or GFP was performed using a flow cytometer with interchangeable optical filters. Parasitaemia was evaluated using different optical filters and, after optimization of optics, the GFP-expressing parasites were sorted and analysed by microscopy after cytospin preparation and by imaging cytometry. Results A new approach to evaluate filter performance in flow cytometry using two-dimensional dot blot was developed. By selecting optical filters with narrow bandpass (BP and maximum position of filter emission close to GFP maximum emission in the FL1 channel (510/20, 512/20 and 517/20; dichroics 502LP and 466LP, AF was markedly decreased and signal-background improve dramatically. Sorting of GFP-expressing parasite populations in infected red blood cells at 90 or 95% purity with these filters resulted in 50-150% increased yield when compared to the standard filter set-up. The purity of the sorted population was confirmed using imaging cytometry and microscopy of cytospin preparations of sorted red blood cells infected with transgenic malaria parasites. Discussion Filter optimization is particularly important for applications where the FP signal and percentage of positive events are relatively low, such as analysis

  10. PHENOTYPING AND SORTING OF MURINE BONE MARROW HAEMATOPOIETIC STEM CELLS USING FLOW CYTOMETRY

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    Kyryk V. M.

    2014-12-01

    Full Text Available To develop a protocol of multiparametric phenotyping and sorting of LSK-subpopulations of hematopoietic stem cells and to determine their relative numbers in the bone marrow of mice was the goal of this research. The modified protocol of multiparametric phenotyping of murine hematopoietic stem cells enable to determine the content of Lin–Sca-1+ c-kit+, Lin–Sca-1+c-kit+flt3+CD150–, Lin–Sca-1+c-kit+flt3+CD150+ and Lin–Sca-1+c-kit+flt3–CD150– subpopulations in bone marrow of FVB mice. It was shown that the dominant population among LSK-cells represents the phenotype Lin–Sca-1+c-kit+flt3–CD150– (57.2 ± 6.8%, which characterizes the short-term hematopoietic stem cells responsible for myelopoiesis. Also the protocol of sorting of murine bone marrow LSK-cells was proposed and its effectiveness for subsequent transplantation in experiments was demonstrated. At repeated phenotyping of sorted cells the purity of Lin–Sca-1+c-kit+ cell fraction was 96.6 ± 1.8% with viability up to 89.6 ± 4.6%.

  11. Index sorting resolves heterogeneous murine hematopoietic stem cell populations

    Science.gov (United States)

    Schulte, Reiner; Wilson, Nicola K.; Prick, Janine C.M.; Cossetti, Chiara; Maj, Michal K.; Gottgens, Berthold; Kent, David G.

    2015-01-01

    Recent advances in the cellular and molecular biology of single stem cells have uncovered significant heterogeneity in the functional properties of stem cell populations. This has prompted the development of approaches to study single cells in isolation, often performed using multiparameter flow cytometry. However, many stem cell populations are too rare to test all possible cell surface marker combinations, and virtually nothing is known about functional differences associated with varying intensities of such markers. Here we describe the use of index sorting for further resolution of the flow cytometric isolation of single murine hematopoietic stem cells (HSCs). Specifically, we associate single-cell functional assay outcomes with distinct cell surface marker expression intensities. High levels of both CD150 and EPCR associate with delayed kinetics of cell division and low levels of differentiation. Moreover, cells that do not form single HSC-derived clones appear in the 7AADdim fraction, suggesting that even low levels of 7AAD staining are indicative of less healthy cell populations. These data indicate that when used in combination with single-cell functional assays, index sorting is a powerful tool for refining cell isolation strategies. This approach can be broadly applied to other single-cell systems, both to improve isolation and to acquire additional cell surface marker information. PMID:26051918

  12. In vivo imaging of alphaherpesvirus infection reveals synchronized activity dependent on axonal sorting of viral proteins.

    Science.gov (United States)

    Granstedt, Andrea E; Bosse, Jens B; Thiberge, Stephan Y; Enquist, Lynn W

    2013-09-10

    A clinical hallmark of human alphaherpesvirus infections is peripheral pain or itching. Pseudorabies virus (PRV), a broad host range alphaherpesvirus, causes violent pruritus in many different animals, but the mechanism is unknown. Previous in vitro studies have shown that infected, cultured peripheral nervous system (PNS) neurons exhibited aberrant electrical activity after PRV infection due to the action of viral membrane fusion proteins, yet it is unclear if such activity occurs in infected PNS ganglia in living animals and if it correlates with disease symptoms. Using two-photon microscopy, we imaged autonomic ganglia in living mice infected with PRV strains expressing GCaMP3, a genetically encoded calcium indicator, and used the changes in calcium flux to monitor the activity of many neurons simultaneously with single-cell resolution. Infection with virulent PRV caused these PNS neurons to fire synchronously and cyclically in highly correlated patterns among infected neurons. This activity persisted even when we severed the presynaptic axons, showing that infection-induced firing is independent of input from presynaptic brainstem neurons. This activity was not observed after infections with an attenuated PRV recombinant used for circuit tracing or with PRV mutants lacking either viral glycoprotein B, required for membrane fusion, or viral membrane protein Us9, required for sorting virions and viral glycoproteins into axons. We propose that the viral fusion proteins produced by virulent PRV infection induce electrical coupling in unmyelinated axons in vivo. This action would then give rise to the synchronous and cyclical activity in the ganglia and contribute to the characteristic peripheral neuropathy.

  13. Cell adhesion and sorting in embryoid bodies derived from N- or E-cadherin deficient murine embryonic stem cells

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    Robert Moore

    2014-01-01

    The primitive endoderm epithelial structure in mouse blastocysts forms following cell differentiation and subsequent sorting, and this two-step process can be reproduced in vitro using an embryoid body model. We found that in the chimeric embryoid bodies consisting of paired wildtype and E-cadherin null ES cells, the wildtype sorted to the center and were enveloped by the less adhesive E-cadherin null cells, in accord with Steinberg's hypothesis. However, wildtype and N-cadherin null ES cells intermixed and did not segregate, a situation that may be explained by Albert Harris' modified principle, which incorporates the unique properties of living cells. Furthermore, in chimeric embryoid bodies composed of N-cadherin and E-cadherin null ES cells, the two weakly interacting cell types segregated but did not envelop one another. Lastly, the most consistent and striking observation was that differentiated cells sorted to the surface and formed an enveloping layer, regardless of the relative cell adhesive affinity of any cell combination, supporting the hypothesis that the ability of the differentiated cells to establish apical polarity is the determining factor in surface sorting and positioning.

  14. Exon loss accounts for differential sorting of Na-K-Cl cotransporters in polarized epithelial cells.

    Science.gov (United States)

    Carmosino, Monica; Giménez, Ignacio; Caplan, Michael; Forbush, Biff

    2008-10-01

    The renal Na-K-Cl cotransporter (NKCC2) is selectively expressed in the apical membranes of cells of the mammalian kidney, where it is the target of the clinically important loop diuretics. In contrast, the "secretory" NKCC1 cotransporter is localized in the basolateral membranes of many epithelia. To identify the sorting signal(s) that direct trafficking of NKCCs, we generated chimeras between the two isoforms and expressed these constructs in polarized renal epithelial cell lines. This analysis revealed an amino acid stretch in NKCC2 containing apical sorting information. The NKCC1 C terminus contains a dileucine motif that constitutes the smallest essential component of its basolateral sorting signal. NKCC1 lacking this motif behaves as an apical protein. Examination of the NKCC gene structure reveals that this dileucine motif is encoded by an additional exon in NKCC1 absent in NKCC2. Phylogenetic analysis of this exon suggests that the evolutionary loss of this exon from the gene encoding the basolateral NKCC1 constitutes a novel mechanism that accounts for the apical sorting of the protein encoded by the NKCC2 gene.

  15. 免疫磁珠分选白血病KG1a细胞中CD34+CD38-干细胞及其特性研究%Isolation and characteristic of CD34 + CD38-stem cells in leukemia cell line KG1a using magnetic activated cell sorting

    Institute of Scientific and Technical Information of China (English)

    王国征; 李慧; 吴远彬; 李达; 贺艳杰; 周雪云

    2013-01-01

    目的 从白血病KGla细胞中分选CD34+ CD38-干细胞并研究其生物学特性.方法 免疫磁珠法分选CD34+ CD38-细胞,流式细胞术分析细胞表面膜抗原、细胞周期,甲基纤维素培养体系观察其克隆性;以HL60、K562、CD34+ CD38+细胞为对照,甲基偶氮唑蓝法检测柔红霉素对CD34+ CD38-细胞的抑制作用;BALB/c裸鼠皮下接种,观察体内成瘤能力.结果 分选的CD34+ CD38-细胞纯度达95%以上,(69.03 +3.25)%处于Go期,克隆形成率为(38.64±2.68)%,明显抵抗柔红霉素;不同浓度柔红霉素作用后,CD34+ CD38-、CD34+ CD38+、HL60、K562细胞的活性差异有统计学意义(F =961.136,P=0.000);CD34+ CD38-在裸鼠皮下成瘤率显著高于CD34+ CD38+细胞(P<0.05).结论 免疫磁珠法分选白血病干细胞简单易行,分选的细胞符合白血病干细胞生物学特性.%Objective To isolate the CD34+ CD38- stem cells from human acute leukemia cell line KGla and to research its biological characteristics. Methods CD34+ CD38- cells were isolated by magnetic activated cell sorting( MACS) ; the cell surface membrane antigens and cell cycle were analyzed by flow cytometry; its clonality was observed with methyl cellulose system. HL60,K562 and D34+ CD38+ cells were selected as control,and the methyl thiazolyl tetrazolium(MTT) assay was taken to observe the depressant effect of rubidomycin to CD34+ CD38- cells. BALB/c nude mice were inoculated subcutaneous-ly to observe the tumorigenicity. Results The purity of CD34+ CD38- cells were above 95% and(69.03 ± 3. 25) % cells in the G0 phase. The cloning efficiency of CD34+ CD38- cells were( 38. 64 ± 2. 68) % , and the CD34+ CD38- cells were obviously resistant to rubidomycin. There were statistic difference of cytoactive among CD34+ CD38-, CD34+ CD38 + , HL60, and K562 cells under giving the same concentrations of rubidomycin circumstances (F = 961. 136,P= 0.000). The tumorigenesis ability of CD34+ CD38- cells in nude mice was

  16. Transcriptional networks in single perivascular cells sorted from human adipose tissue reveal a hierarchy of mesenchymal stem cells.

    Science.gov (United States)

    Hardy, W Reef; Moldovan, Nicanor I; Moldovan, Leni; Livak, Kenneth J; Datta; Goswami, Chirayu; Corselli, Mirko; Traktuev, Dmitry O; Murray, Iain R; Péault, Bruno; March, Keith

    2017-02-24

    Adipose tissue is a rich source of multipotent mesenchymal stem-like cells, located in the perivascular niche. Based on their surface markers, these have been assigned to two main categories: CD34+CD31-CD45-CD146- cells (adventitial stromal/stem cells, ASCs), and CD146+CD31-CD34-CD45- cells (pericytes, PCs). These populations display heterogeneity of unknown significance. We hypothesized that aldehyde dehydrogenase (ALDH) activity, a functional marker of primitivity, could help to better define ASC and PC subclasses. To this end, the stromal vascular fraction from a human lipoaspirate was simultaneously stained with fluorescent antibodies to CD31, CD45, CD34, and CD146 antigens and the ALDH substrate Aldefluor®, then sorted by FACS. Individual ASCs (n=67) and PCs (n=73) selected from the extremities of the ALDH-staining spectrum were transcriptionally profiled by Fluidigm single-cell quantitative PCR for a predefined set (n=429) of marker genes. To these single-cell data, we applied differential expression and principal component and clustering analysis, as well as an original gene co-expression network reconstruction algorithm. Despite the stochasticity at the single-cell level, covariation gene expression analysis yielded multiple network connectivity parameters suggesting that these perivascular progenitor cell subclasses possess the following order of maturity: i) ALDH(br) ASC (most primitive); ii) ALDH(dim) ASC; iii) ALDH(br) PC; iv) ALDH(dim) PC (least primitive). This order was independently supported by specific combinations of class-specific expressed genes and further confirmed by the analysis of associated signaling pathways. In conclusion, single-cell transcriptional analysis of four populations isolated from fat by surface markers and enzyme activity suggests a developmental hierarchy among perivascular mesenchymal stem cells supported by markers and co-expression networks. This article is protected by copyright. All rights reserved.

  17. echinus, required for interommatidial cell sorting and cell death in the Drosophila pupal retina, encodes a protein with homology to ubiquitin-specific proteases

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    Gorski Sharon M

    2007-07-01

    Full Text Available Abstract Background Programmed cell death is used to remove excess cells between ommatidia in the Drosophila pupal retina. This death is required to establish the crystalline, hexagonal packing of ommatidia that characterizes the adult fly eye. In previously described echinus mutants, interommatidial cell sorting, which precedes cell death, occurred relatively normally. Interommatidial cell death was partially suppressed, resulting in adult eyes that contained excess pigment cells, and in which ommatidia were mildly disordered. These results have suggested that echinus functions in the pupal retina primarily to promote interommatidial cell death. Results We generated a number of new echinus alleles, some likely null mutants. Analysis of these alleles provides evidence that echinus has roles in cell sorting as well as cell death. echinus encodes a protein with homology to ubiquitin-specific proteases. These proteins cleave ubiquitin-conjugated proteins at the ubiquitin C-terminus. The echinus locus encodes multiple splice forms, including two proteins that lack residues thought to be critical for deubiquitination activity. Surprisingly, ubiquitous expression in the eye of versions of Echinus that lack residues critical for ubiquitin specific protease activity, as well as a version predicted to be functional, rescue the echinus loss-of-function phenotype. Finally, genetic interactions were not detected between echinus loss and gain-of-function and a number of known apoptotic regulators. These include Notch, EGFR, the caspases Dronc, Drice, Dcp-1, Dream, the caspase activators, Rpr, Hid, and Grim, the caspase inhibitor DIAP1, and Lozenge or Klumpfuss. Conclusion The echinus locus encodes multiple splice forms of a protein with homology to ubiquitin-specific proteases, but protease activity is unlikely to be required for echinus function, at least when echinus is overexpressed. Characterization of likely echinus null alleles and genetic interactions

  18. An efficient method of sorting liver stem cells by using immuno-magnetic microbeads

    Institute of Scientific and Technical Information of China (English)

    Yu-Fei He; Yin-Kun Liu; Dong-Mei Gao; Jun Chen; Peng-Yuan Yang

    2006-01-01

    AIM: To develop a method to isolate liver stem cells fast and efficiently.METHODS: Fetal mouse liver cells were characterized by cell surface antigens (c-Kit and CD45/TER119) using flow cytometry. The candidate liver stem cells were sorted by using immuno-magnetic microbeads and identified by clone-forming culture, RT-PCR and immunofluorescence assays.RESULTS: The c-Kit-(CD45/TER119)-cell population with 97.9% of purity were purified by immuno-magnetic microbeads at one time. The yield of this separation was about 6% of the total sorting cells and the cell viability was above 98%. When cultured in vitro these cells had high clone-forming and self-renewing ability and expressed markers of hepatocytes and bile duct cells.Functionally mature hepatocytes were observed after 21 d of culture.CONCLUSION: This method offers an excellent tool for the enrichment of liver stem cells with high purity and viability, which could be used for further studies. It is fast, efficient, simple and not expensive.

  19. Continuous high throughput molecular adhesion based cell sorting using ridged microchannels

    Science.gov (United States)

    Tasadduq, Bushra; Wang, Gonghao; Alexeev, Alexander; Sarioglu, Ali Fatih; Sulchek, Todd

    2016-11-01

    Cell molecular interactions govern important physiological processes such as stem cell homing, inflammation and cancer metastasis. But due to a lack of effective separation technologies selective to these interactions it is challenging to specifically sort cells. Other label free separation techniques based on size, stiffness and shape do not provide enough specificity to cell type, and correlation to clinical condition. We propose a novel microfluidic device capable of high throughput molecule dependent separation of cells by flowing them through a microchannel decorated with molecule specific coated ridges. The unique aspect of this sorting design is the use of optimized gap size which is small enough to lightly squeeze the cells while flowing under the ridged part of the channel to increase the surface area for interaction between the ligand on cell surface and coated receptor molecule but large enough so that biomechanical markers, stiffness and viscoelasticity, do not dominate the cell separation mechanism. We are able to separate Jurkat cells based on its expression of PSGL-1ligand using ridged channel coated with P selectin at a flow rate of 0.045ml/min and achieve 2-fold and 5-fold enrichment of PSGL-1 positive and negative Jurkat cells respectively.

  20. Functional single-cell analyses: flow cytometry and cell sorting of microbial populations and communities.

    Science.gov (United States)

    Müller, Susann; Nebe-von-Caron, Gerhard

    2010-07-01

    The still poorly explored world of microbial functioning is about to be uncovered by a combined application of old and new technologies. Bacteria, especially, are still in the dark with respect to their phylogenetic affiliations as well as their metabolic capabilities and functions. However, with the advent of sophisticated flow cytometric and cell sorting technologies in microbiological labs, there is now the possibility to gain this knowledge at the single-cell level without cumbersome cultivation approaches. Cytometry also facilitates the understanding of physiological diversity in seemingly likewise acting populations. Both individuality and diversity lead to the complex and concerted actions of microbial consortia. This review provides an overview of the state of the art in the field. It deals with the handling of microorganisms from the very beginning (i.e. sampling, and detachment and fixation procedures) and goes on to discuss the pitfalls and problems in analysing cells without any further treatment. If information cannot be gained by specific staining procedures, phylogenetic technologies, transcriptomic and proteomic approaches may be options for achieving advanced insights. All in all, flow cytometry will be a mediator technology to gain a deeper insight into the heterogeneity of populations and the functioning of microbial communities.

  1. 人外周血树突状细胞体外稳定培养方法的建立及与磁珠法的比较%Establishment of in vitro stable culture of human peripheral blood dendritic cells and its comparation with magnetic activated cell sorting

    Institute of Scientific and Technical Information of China (English)

    单骄宇; 刘弓伯; 吐尔洪江·吐尔逊; 张雪; 阿尔孜古丽·吐逊; 林仁勇; 温浩

    2010-01-01

    Objective To establish a economic and stable method to induce and culture dendritic cells (DCs) from peripheral blood of human being, and compare with the magnetic activated cell sorting. Methods Monocytes were isolated from health donors peripheral blood mononuclear cells(PBMC) by density gradient separation,cultured and compared with that of cells isolated by the magnetic activated cell sorting or adherent culture,respectively. PBMC were cultured with recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) and recombinant human interleukin-4(rhIL-4) for 6 days to induce the growth of DCs. Morphological changes was observed under inverted microscope. Meanwhile, cell viability was tested at the 3rd, 5th, 6th day,respectively. The phenotypes, like CD14, CDla, HLA-DR were analyzed with flow cytometry after PBMC were adherent cultured for 1, 2, 5 h. After adding human recombinant cytokines, the phenotypes of acquired cells surface markers, CD14, CD1a, CD86, CD83 and HLA-DR would be detected and compared with flow cytometry. T cells proliferating activity was determined by allogeneic mixed lymphocyte reaction in vitro. Results After adherent culture for 2 h, the acquired DCs showed typical morphology. Cell viability was decreased at days 5th, 6th[(53.333 ±5.774)%,(38.333 ± 7.638)%] than that at day of 3rd[(68.667 ± 3.215)%, all P < 0.05] with the magnetic activated cell sorting, but with adherent culture method, the difference was not statistically significant (F = 0.737,P> 0.05) at days of 3rd, 5th, 6th[(92.667 ± 3.055)%,(94.000 ± 1.000)%,(94.667 ± 1.528)%]. Moreover,compared with the magnetic activated cell sorting, there were differences in cell viability of adherent culture method at days of 3rd, 5th, 6th(t = 9.374, 12.021,12.527, all P < 0.05). Before and after using the magnetic activated cell sorting, the expression of CD14 were (32.457 ± 12.351) %, (41.914 ± 14.858)%, respectively. The difference was not statistically

  2. Dipeptidyl peptidase IV is sorted to the secretory granules in pancreatic islet A-cells

    DEFF Research Database (Denmark)

    Poulsen, Mona Dam; Hansen, Gert Helge; Dabelsteen, Erik

    1993-01-01

    Dipeptidyl peptidase IV (DP IV:EC 3.4.14.5) was localized in endocrine cells of pig pancreas by immunohistochemical and enzyme histochemical methods. Immunolight microscopy with both monoclonal and polyclonal antibodies demonstrated DP IV immunoreactivity in cells located in the peripheral part...... labeling using a monoclonal glucagon antibody as the second primary antibody. These results show that DP IV is sorted to secretory granules in the pig pancreatic islet A-cells. Furthermore, this secretory granule enzyme, as opposed to intestinal brush border DP IV, is suggested to be a soluble protein...

  3. Sorting of Streptomyces cell pellets using a complex object parametric analyzer and sorter.

    Science.gov (United States)

    Petrus, Marloes L C; van Veluw, G Jerre; Wösten, Han A B; Claessen, Dennis

    2014-02-13

    Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these organisms form networks of interconnected hyphae, known as pellets, which are heterogeneous in size. Here we describe a method to analyze and sort mycelial pellets using a Complex Object Parametric Analyzer and Sorter (COPAS). Detailed instructions are given for the use of the instrument and the basic statistical analysis of the data. We furthermore describe how pellets can be sorted according to user-defined settings, which enables downstream processing such as the analysis of the RNA or protein content. Using this methodology the mechanism underlying heterogeneous growth can be tackled. This will be instrumental for improving streptomycetes as a cell factory, considering the fact that productivity correlates with pellet size.

  4. Measuring and sorting cell populations expressing isospectral fluorescent proteins with different fluorescence lifetimes.

    Directory of Open Access Journals (Sweden)

    Bryan Sands

    Full Text Available Study of signal transduction in live cells benefits from the ability to visualize and quantify light emitted by fluorescent proteins (XFPs fused to different signaling proteins. However, because cell signaling proteins are often present in small numbers, and because the XFPs themselves are poor fluorophores, the amount of emitted light, and the observable signal in these studies, is often small. An XFP's fluorescence lifetime contains additional information about the immediate environment of the fluorophore that can augment the information from its weak light signal. Here, we constructed and expressed in Saccharomyces cerevisiae variants of Teal Fluorescent Protein (TFP and Citrine that were isospectral but had shorter fluorescence lifetimes, ∼ 1.5 ns vs ∼ 3 ns. We modified microscopic and flow cytometric instruments to measure fluorescence lifetimes in live cells. We developed digital hardware and a measure of lifetime called a "pseudophasor" that we could compute quickly enough to permit sorting by lifetime in flow. We used these abilities to sort mixtures of cells expressing TFP and the short-lifetime TFP variant into subpopulations that were respectively 97% and 94% pure. This work demonstrates the feasibility of using information about fluorescence lifetime to help quantify cell signaling in living cells at the high throughput provided by flow cytometry. Moreover, it demonstrates the feasibility of isolating and recovering subpopulations of cells with different XFP lifetimes for subsequent experimentation.

  5. Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases

    OpenAIRE

    Arildo Nerys-Junior; Costa, Lendel C.; Braga-Dias,Luciene P.; Márcia Oliveira; Rossi,Átila D.; Rodrigo Delvecchio da Cunha; Gonçalves,Gabriel S.; Amilcar Tanuri

    2014-01-01

    Engineered nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) are one of the most promising tools for modifying genomes. These site-specific enzymes cause double- strand breaks that allow gene disruption or gene insertion, thereby facilitating genetic manipulation. The major problem associated with this approach is the labor-intensive procedures required to screen and confirm the cellular modification by nucleases. In this work, we produc...

  6. One-step fabrication of 3D silver paste electrodes into microfluidic devices for enhanced droplet-based cell sorting

    Directory of Open Access Journals (Sweden)

    Lang Rao

    2015-05-01

    Full Text Available 3D microelectrodes are one-step fabricated into a microfluidic droplet separator by filling conductive silver paste into PDMS microchambers. The advantages of 3D silver paste electrodes in promoting droplet sorting accuracy are systematically demonstrated by theoretical calculation, numerical simulation and experimental validation. The employment of 3D electrodes also helps to decrease the droplet sorting voltage, guaranteeing that cells encapsulated in droplets undergo chip-based sorting processes are at better metabolic status for further potential cellular assays. At last, target droplet containing single cell are selectively sorted out from others by an appropriate electric pulse. This method provides a simple and inexpensive alternative to fabricate 3D electrodes, and it is expected our 3D electrode-integrated microfluidic droplet separator platform can be widely used in single cell operation and analysis.

  7. Flow-cytometry and cell sorting: an efficient approach to investigate productivity and cell physiology in mammalian cell factories.

    Science.gov (United States)

    Kumar, Niraj; Borth, Nicole

    2012-03-01

    The performance of cell lines used for the production of biotherapeutic proteins typically depends on the number of cells in culture, their specific growth rate, their viability and the cell specific productivity (qP). Therefore both cell line development and process development are trying to (a) improve cell proliferation to reduce lag-phase and achieve high number of cells; (b) delay cell death to prolong the production phase and improve culture longevity; (c) and finally, increase qP. All of these factors, when combined in an optimised process, concur to increase the final titre and yield of the recombinant protein. As cellular performance is at the centre of any improvement, analysis methods that enable the characterisation of individual cells in their entirety can help in identifying cell types and culture conditions that perform exceptionally well. This observation of cells and their complexity is reflected by the term "cytomics" and flow cytometry is one of the methods used for this purpose. With its ability to analyse the distribution of physiological properties within a population and to isolate rare outliers with exceptional properties, flow cytometry ideally complements other methods used for optimisation, including media design and cell engineering. In the present review we describe approaches that could be used, directly or indirectly, to analyse and sort cellular phenotypes characterised by improved growth behaviour, reduced cell death or high qP and outline their potential use for cell line and process optimisation.

  8. SorLA Controls Neurotrophic Activity by Sorting of GDNF and Its Receptors GFRα1 and RET

    DEFF Research Database (Denmark)

    Glerup, Simon; Lume, Maria; Olsen, Ditte;

    2013-01-01

    Glial cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor that has reached clinical trials for Parkinson's disease. GDNF binds to its coreceptor GFRα1 and signals through the transmembrane receptor tyrosine kinase RET, or RET independently through NCAM or syndecan-3....... Whereas the GDNF signaling cascades are well described, cellular turnover and trafficking of GDNF and its receptors remain poorly characterized. Here, we find that SorLA acts as sorting receptor for the GDNF/GFRα1 complex, directing it from the cell surface to endosomes. Through this mechanism, GDNF...... is targeted to lysosomes and degraded while GFRα1 recycles, creating an efficient GDNF clearance pathway. The SorLA/GFRα1 complex further targets RET for endocytosis but not for degradation, affecting GDNF-induced neurotrophic activities. SorLA-deficient mice display elevated GDNF levels, altered dopaminergic...

  9. Identification and purification of classical Hodgkin cells from lymph nodes by flow cytometry and flow cytometric cell sorting.

    Science.gov (United States)

    Fromm, Jonathan R; Kussick, Steven J; Wood, Brent L

    2006-11-01

    We demonstrate the feasibility of using flow cytometry (FC) to identify the Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (CHL). Initial flow cytometric studies of the HRS cell line L1236 demonstrated potentially useful antigens for identifying HRS cells. L1236 cells spontaneously bound normal T cells, analogous to the T-cell rosetting of HRS cells seen in tissue sections of CHL, but these interactions could be blocked by using a cocktail of unlabeled antibodies to 4 adhesion molecules. Among 27 lymph nodes involved by CHL, FC enabled HRS cells to be identified in 89%, whereas none of 29 non-CHL neoplasms or 23 reactive lymph nodes demonstrated HRS populations. Of the CHL cases, 82% demonstrated interactions between HRS cells and T cells that could be disrupted with blocking antibodies. Flow cytometric cell sorting experiments demonstrated typical HRS cytomorphologic features among the purified cells. FC may offer an alternative to immunohistochemical analysis in confirming the diagnosis of CHL in certain cases, and, through cell sorting, it provides a means of rapidly isolating pure HRS cells.

  10. The functional architecture of the human body: assessing body representation by sorting body parts and activities.

    Science.gov (United States)

    Bläsing, Bettina; Schack, Thomas; Brugger, Peter

    2010-05-01

    We investigated mental representations of body parts and body-related activities in two subjects with congenitally absent limbs (one with, the other without phantom sensations), a wheelchair sports group of paraplegic participants, and two groups of participants with intact limbs. To analyse mental representation structures, we applied Structure Dimensional Analysis. Verbal labels indicating body parts and related activities were presented in randomized lists that had to be sorted according to a hierarchical splitting paradigm. Participants were required to group the items according to whether or not they were considered related, based on their own body perception. Results of the groups of physically intact and paraplegic participants revealed separate clusters for the lower body, upper body, fingers and head. The participant with congenital phantom limbs also showed a clear separation between upper and lower body (but not between fingers and hands). In the participant without phantom sensations of the absent arms, no such modularity emerged, but the specific practice of his right foot in communication and daily routines was reflected. Sorting verbal labels of body parts and activities appears a useful method to assess body representation in individuals with special body anatomy or function and leads to conclusions largely compatible with other assessment procedures.

  11. Improved transgene expression in doxycycline-inducible embryonic stem cells by repeated chemical selection or cell sorting

    Directory of Open Access Journals (Sweden)

    Renáta Bencsik

    2016-09-01

    Full Text Available Transgene-mediated programming is a preeminent strategy to direct cellular identity. To facilitate cell fate switching, lineage regulating genes must be efficiently and uniformly induced. However, gene expression is often heterogeneous in transgenic systems. Consistent with this notion, a non-uniform reporter gene expression was detected in our doxycycline (DOX-regulated, murine embryonic stem (ES cell clones. Interestingly, a significant fraction of cells within each clone failed to produce any reporter signals upon DOX treatment. We found that the majority of these non-responsive cells neither carry reporter transgene nor geneticin/G418 resistance. This observation suggested that our ES cell clones contained non-recombined cells that survived the G418 selection which was carried out during the establishment of these clones. We successfully eliminated most of these corrupted cells with repeated chemical (G418 selection, however, even after prolonged G418 treatments, a few cells remained non-responsive due to epigenetic silencing. We found that cell sorting has been the most efficient approach to select those cells which can uniformly and stably induce the integrated transgene in this ES cell based platform. Together, our data revealed that post-cloning chemical re-selection or cell sorting strongly facilitate the production of ES cell lines with a uniform transgene induction capacity.

  12. Efficient Parallel Sorting for Migrating Birds Optimization When Solving Machine-Part Cell Formation Problems

    Directory of Open Access Journals (Sweden)

    Ricardo Soto

    2016-01-01

    Full Text Available The Machine-Part Cell Formation Problem (MPCFP is a NP-Hard optimization problem that consists in grouping machines and parts in a set of cells, so that each cell can operate independently and the intercell movements are minimized. This problem has largely been tackled in the literature by using different techniques ranging from classic methods such as linear programming to more modern nature-inspired metaheuristics. In this paper, we present an efficient parallel version of the Migrating Birds Optimization metaheuristic for solving the MPCFP. Migrating Birds Optimization is a population metaheuristic based on the V-Flight formation of the migrating birds, which is proven to be an effective formation in energy saving. This approach is enhanced by the smart incorporation of parallel procedures that notably improve performance of the several sorting processes performed by the metaheuristic. We perform computational experiments on 1080 benchmarks resulting from the combination of 90 well-known MPCFP instances with 12 sorting configurations with and without threads. We illustrate promising results where the proposal is able to reach the global optimum in all instances, while the solving time with respect to a nonparallel approach is notably reduced.

  13. 磁激活细胞分选术联合混合抗体提高模拟恶性腹水中游离癌细胞检出效率的分析%Magnetic activated cell sorting combined with a panel of monoclonal antibodies in detecting free cancer cells in analogue malignant ascites

    Institute of Scientific and Technical Information of China (English)

    王晓蕾; 陈锡美; 黄志刚; 王韶英

    2008-01-01

    背景磁激活细胞分选术(magnetic activated cell sorting,MACS)是一种新的免疫磁性分离技术.其原理是基于抗体对抗原的特异性识别,将50 nm磁性微珠直接或者间接耦联在抗体上,与有相应抗原表达的细胞相连,在高强度、高梯度磁场中达到细胞磁性分离的目的.该法具有分离纯度和回收率均较高的优势,也能分离出体液中存在的少量肿瘤细胞.目的评价MACS联合一组肿瘤细胞标志物的方法对提高模拟恶性腹水中游离癌细胞检出效率的作用.方法 选择5种与恶性腹水病因有关的肿瘤细胞株作为研究对象,采用免疫荧光反应和流式细胞仪(FCM)检测上皮相关抗原(epithlial-related antigen,EpCAM)、CA125、癌胚抗原和TAG-72共4种单克隆抗体及其混合抗体在各肿瘤细胞的表达.将肿瘤细胞以不同比例掺入单个核细胞中模拟恶性腹水细胞成分,与联合单个抗体进行分选对比,观察MACS术联合混合抗体分选肿瘤细胞的效率.结果 FCM结果表明,混合抗体在5种肿瘤细胞的阳性表达率均高于4种抗体单独反应的阳性率.MACS术联合混合抗体检出模拟恶性腹水中肿瘤细胞的得率比联合单个抗体的得率要高,其中以2种胃癌细胞和结肠癌细胞的平均得率提高较多(分别为69.18%±20.84%比45.23%±11.54%、78.75%±15.42%比59.73%±16.64%和85.63%±12.30%比76.88%±8.65%),卵巢癌细胞次之(32.49%±3.58%比31.79%±4.82%),肝癌细胞最少(11.78%±0.43%比7.16%±0.46%).结论 与联合单个抗体进行分选相比,应用MACS术联合一组混合抗体的方法可有效提高恶性腹水中游离癌细胞的检出效率,尤其对胃肠道肿瘤所致的恶性腹水有潜在的临床应用价值.%Background Magnetic activated cell sorting(MACS)is a new immunomagnetic separation technique.It's principle is based on the specialized recognition between the antibody and antigen.When directed or indirected coupled with the 50 nm

  14. Numerical study on the complete blood cell sorting using particle tracing and dielectrophoresis in a microfluidic device

    Science.gov (United States)

    Ali, Haider; Park, Cheol Woo

    2016-11-01

    In this study, a numerical model of a microfluidic device with particle tracing and dielectrophoresis field-flow fractionation was employed to perform a complete and continuous blood cell sorting. A low voltage was applied to electrodes to separate the red blood cells, white blood cells, and platelets based on their cell size. Blood cell sorting and counting were performed by evaluating the cell trajectories, displacements, residence times, and recovery rates in the device. A novel numerical technique was used to count the number of separated blood cells by estimating the displacement and residence time of the cells in a microfluidic device. For successful blood cell sorting, the value of cells displacement must be approximately equal to or higher than the corresponding maximum streamwise distance. The study also proposed different outlet designs to improve blood cell separation. The basic outlet design resulted in a higher cells recovery rate than the other outlets design. The recovery rate decreased as the number of inlet cells and flow rates increased because of the high particle-particle interactions and collisions with walls. The particle-particle interactions significantly affect blood cell sorting and must therefore be considered in future work.

  15. Sorting of cells of the same size, shape, and cell cycle stage for a single cell level assay without staining

    Directory of Open Access Journals (Sweden)

    Yomo Tetsuya

    2006-06-01

    Full Text Available Abstract Background Single-cell level studies are being used increasingly to measure cell properties not directly observable in a cell population. High-performance data acquisition systems for such studies have, by necessity, developed in synchrony. However, improvements in sample purification techniques are also required to reveal new phenomena. Here we assessed a cell sorter as a sample-pretreatment tool for a single-cell level assay. A cell sorter is routinely used for selecting one type of cells from a heterogeneous mixture of cells using specific fluorescence labels. In this case, we wanted to select cells of exactly the same size, shape, and cell-cycle stage from a population, without using a specific fluorescence label. Results We used four light scatter parameters: the peak height and area of the forward scatter (FSheight and FSarea and side scatter (SSheight and SSarea. The rat pheochromocytoma PC12 cell line, a neuronal cell line, was used for all experiments. The living cells concentrated in the high FSarea and middle SSheight/SSarea fractions. Single cells without cell clumps were concentrated in the low SS and middle FS fractions, and in the higher FSheight/FSarea and SSheight/SSarea fractions. The cell populations from these viable, single-cell-rich fractions were divided into twelve subfractions based on their FSarea-SSarea profiles, for more detailed analysis. We found that SSarea was proportional to the cell volume and the FSarea correlated with cell roundness and elongation, as well as with the level of DNA in the cell. To test the method and to characterize the basic properties of the isolated single cells, sorted cells were cultured in separate wells. The cells in all subfractions survived, proliferated and differentiated normally, suggesting that there was no serious damage. The smallest, roundest, and smoothest cells had the highest viability. There was no correlation between proliferation and differentiation. NGF increases

  16. Continuous-flow microfluidic blood cell sorting for unprocessed whole blood using surface-micromachined microfiltration membranes.

    Science.gov (United States)

    Li, Xiang; Chen, Weiqiang; Liu, Guangyu; Lu, Wei; Fu, Jianping

    2014-07-21

    White blood cells (WBCs) constitute about 0.1% of the blood cells, yet they play a critical role in innate and adaptive immune responses against pathogenic infections, allergic conditions, and malignancies and thus contain rich information about the immune status of the body. Rapid isolation of WBCs directly from whole blood is a prerequisite for any integrated immunoassay platform designed for examining WBC phenotypes and functions; however, such functionality is still challenging for blood-on-a-chip systems, as existing microfluidic cell sorting techniques are inadequate for efficiently processing unprocessed whole blood on chip with concurrent high throughput and cell purity. Herein we report a microfluidic chip for continuous-flow isolation and sorting of WBCs from whole blood with high throughput and separation efficiency. The microfluidic cell sorting chip leveraged the crossflow filtration scheme in conjunction with a surface-micromachined poly(dimethylsiloxane) (PDMS) microfiltration membrane (PMM) with high porosity. With a sample throughput of 1 mL h(-1), the microfluidic cell sorting chip could recover 27.4 ± 4.9% WBCs with a purity of 93.5 ± 0.5%. By virtue of its separation efficiency, ease of sample recovery, and high throughput enabled by its continuous-flow operation, the microfluidic cell sorting chip holds promise as an upstream component for blood sample preparation and analysis in integrated blood-on-a-chip systems.

  17. Principles and applications of flow cytometry and cell sorting in companion animal medicine.

    Science.gov (United States)

    Wilkerson, Melinda J

    2012-01-01

    Flow cytometry measures multiple characteristic of single cells using light scatter properties and fluorescence properties of fluorescent probes with specificity to cellular constituents. The use of flow cytometry in the veterinary clinical laboratory has become more routine in veterinary diagnostic laboratories and institutions (http://www.vet.k-state.edu/depts/dmp/service/immunology/index.htm), and reference laboratories. The most common applications in small animal medicine includes quantitation of erythrocytes and leukocytes in automated hematology instruments, detection of antibodies to erythrocytes and platelets in cases of immune-mediated diseases, immunophenotyping of leukocytes and lymphocytes in immunodeficiency syndromes, or leukemias and lymphomas. DNA content analysis to identify aneuploidy or replicating cells in tumor preparations has not gained routine acceptance because of the variability of prognostic results. Other applications including cell sorting and multiplexing using microspheres are potential assays of the future once they become validated and the instrumentation footprint becomes more and more compact, less expensive, and easier to use.

  18. Multiparametric analysis, sorting, and transcriptional profiling of plant protoplasts and nuclei according to cell type.

    Science.gov (United States)

    Galbraith, David W; Janda, Jaroslav; Lambert, Georgina M

    2011-01-01

    Flow cytometry has been employed for the analysis of higher plants for approximately the last 30 years. For the angiosperms, ∼500,000 species, itself a daunting number, parametric measurements enabled through the use of flow cytometers started with basic descriptors of the individual cells and their contents, and have both inspired the development of novel cytometric methods that subsequently have been applied to organisms within other kingdoms of life, and adopted cytometric methods devised for other species, particularly mammals. Higher plants offer unique challenges in terms of flow cytometric analysis, notably the facts that their organs and tissues are complex three-dimensional assemblies of different cell types, and that their individual cells are, in general, larger than those of mammals.This chapter provides an overview of the general types of parametric measurement that have been applied to plants, and provides detailed methods for selected examples based on the plant model Arabidopsis thaliana. These illustrate the use of flow cytometry for the analysis of protoplasts and nuclear DNA contents (genome size and the cell cycle). These are further integrated with measurements focusing on specific cell types, based on transgenic expression of Fluorescent Proteins (FPs), and on analysis of the spectrum of transcripts found within protoplasts and nuclei. These measurements were chosen in particular to illustrate, respectively, the issues encountered in the flow analysis and sorting of large biological cells, typified by protoplasts; how to handle flow analyses under conditions that require processing of large numbers of samples in which the individual samples contain only a very small minority of objects of interest; and how to deal with exceptionally small amounts of RNA within the sorted samples.

  19. Characteristics of calves produced with sperm sexed by flow cytometry/cell sorting.

    Science.gov (United States)

    Tubman, L M; Brink, Z; Suh, T K; Seidel, G E

    2004-04-01

    The objectives of this study were to determine whether calves produced by sexed sperm differed from controls and to what extent the sex ratio of calves was altered by the sexing procedure. Data were collected from 1,169 calves produced from sperm sexed by flow cytometry/cell sorting after staining with Hoechst 33342, and 793 calves produced from control sperm during breeding trials between 1997 and 2001. Least squares ANOVA were completed using factors of treatment (sexed vs. control sperm), 19 management groups from 13 field trials, and calf sex. Responses analyzed include gestation length, birth weight, calving ease, calf vigor, weaning weight, abortion rate, and death rates (neonatal and through weaning). No significant difference was observed for any response due to treatment or treatment interactions (P > 0.10). Therefore, calves produced from sexed sperm grew and developed normally both pre- and postnatally. A neurological disorder was observed in four control calves and one sexed calf from one farm. No gross anatomical abnormalities were reported for any calves in the study. Differences were observed for all responses among management groups (P Flow cytometry/cell sorting can be used to preselect sex of calves safely with approximately 90% accuracy.

  20. Approaches for cytogenetic and molecular analyses of small flow-sorted cell populations from childhood leukemia bone marrow samples

    DEFF Research Database (Denmark)

    Obro, Nina Friesgaard; Madsen, Hans O.; Ryder, Lars Peter;

    2011-01-01

    defined cell populations with subsequent analyses of leukemia-associated cytogenetic and molecular marker. The approaches described here optimize the use of the same tube of unfixed, antibody-stained BM cells for flow-sorting of small cell populations and subsequent exploratory FISH and PCR-based analyses....

  1. Isolation of Human Induced Pluripotent Stem Cell-Derived Dopaminergic Progenitors by Cell Sorting for Successful Transplantation

    Directory of Open Access Journals (Sweden)

    Daisuke Doi

    2014-03-01

    Full Text Available Human induced pluripotent stem cells (iPSCs can provide a promising source of midbrain dopaminergic (DA neurons for cell replacement therapy for Parkinson’s disease. However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells. Here, we show that human iPSC-derived DA progenitor cells can be efficiently isolated by cell sorting using a floor plate marker, CORIN. We induced DA neurons using scalable culture conditions on human laminin fragment, and the sorted CORIN+ cells expressed the midbrain DA progenitor markers, FOXA2 and LMX1A. When transplanted into 6-OHDA-lesioned rats, the CORIN+ cells survived and differentiated into midbrain DA neurons in vivo, resulting in significant improvement of the motor behavior, without tumor formation. In particular, the CORIN+ cells in a NURR1+ cell-dominant stage exhibited the best survival and function as DA neurons. Our method is a favorable strategy in terms of scalability, safety, and efficiency and may be advantageous for clinical application.

  2. Strategies for immunophenotyping and purifying classical Hodgkin lymphoma cells from lymph nodes by flow cytometry and flow cytometric cell sorting.

    Science.gov (United States)

    Fromm, Jonathan R; Wood, Brent L

    2012-07-01

    Flow cytometry is an established technique to immunophenotype hematopoietic neoplasms. While the diagnosis of classical Hodgkin lymphoma (CHL) has commonly been made using paraffin sections, we have recently demonstrated that the neoplastic Hodgkin and Reed-Sternberg (HRS) cells of CHL can be identified by flow cytometry. Using 6- and 9-color flow cytometric assays, CHL can be immunophenotyped with 85-90% sensitivity and nearly 100% specificity. Analysis of this data requires using established gating strategies to help in the identification of putative HRS cell populations. Interestingly, HRS cells bind to reactive T cells (HRS-T cell rosetting) and this phenomenon can be identified and utilized diagnostically by flow cytometry. In addition, the reactive T cells of CHL show characteristic immunophenotypic changes by flow cytometry and these changes can suggest a diagnosis of CHL. Finally, these principles can be employed to rapidly purify HRS cells using flow cytometric cell sorting. This manuscript provides experimental protocols for immunophenotyping CHL by flow cytometry as well as purifying the HRS cells via flow cytometric cell sorting.

  3. 免疫磁性活化细胞分选方法优化及纯化后细胞的生物学特性检测%Optimization of magnetic activated cell sorting and the biological characteristics of isolated cells

    Institute of Scientific and Technical Information of China (English)

    耿珊; 张琛; 刘俊; 刘典锋; 周玥; 王亚平

    2012-01-01

    目的:改良常规免疫磁性活化细胞分选(MACS)的分离纯化方法,提高分选后细胞的纯度并确保细胞仍具有良好活性及功能.方法:以干细胞抗原-1(Sca-1)标记的Sca-1+造血干/祖细胞(Sca-1+ HSC/HPC)为例,分别用改良后和常规MACS法分选出小鼠骨髓细胞Sca-1+细胞,流式细胞术检测两种分选方法获得Sca-1+细胞纯度;计算阳性细胞回收率;台酚蓝染色检测分选细胞存活百分率;CCK-8检测细胞增殖,混合造血祖细胞( CFU-Mix)体外培养评价分选Sca-1+细胞分化能力.结果:改良MACS法分选获得的Sca-1+细胞纯度达93%以上(常规MACS法仅为87%),阳性细胞的回收率可达73%(常规法仅为62.3%);细胞存活率、细胞增殖和CFU-Mix集落形成能力两种分选方法无明显差别.结论:改良法能明显提高细胞分选纯度及回收率,细胞活性和功能保持良好,值得各种细胞免疫磁性分选参考采用.%AIM: To optimize the traditional magnetic activated cell sorting (MACS) so as to enhance the purification and keep the viability of cells after separation. METHODS; Take stem cell antigen-1 labeled hemopoietic stem/ progenitor cells (Sca-1+ HSC/HPC) for example. The traditional MACS and the optimized MACS were applied to obtain the Sca-1 + HSC/HPC from mouse bone marrow respectively. The purifications of Sca-1 + cells from two groups were tested by flow cytometry; The survival rates of Sca-1 + cells from two groups were detected by trypan blue dye; The proliferation of Sca-1 + cells was detected by cell counting kit-8( CCK-8); the differentiation capacity of Sca-1+ cells was measured by CFU-Mix. RESULTS: The purification of Sca-1 + cells was up to 93% by the optimized MACS compared to 87% by the traditional one-, the recovery rate of positive cells was 73% by the optimized MACS compared to 62.3% by the traditional one; there was no statistical difference in the viability and proliferation of Sca-1+ cells and the capacity

  4. Flow sorting in aquatic ecology

    OpenAIRE

    Marcus Reckermann

    2000-01-01

    Flow sorting can be a very helpful tool in revealing phytoplankton and bacterial community structure and elaborating specific physiological parameters of isolated species. Droplet sorting has been the most common technique. Despite the high optical and hydro-dynamic stress for the cells to be sorted, many species grow in culture subsequent to sorting. To date, flow sorting has been applied to post-incubation separation in natural water samples to account for group-specific physiological param...

  5. Using injection molding and reversible bonding for easy fabrication of magnetic cell trapping and sorting devices

    Science.gov (United States)

    Royet, David; Hériveaux, Yoann; Marchalot, Julien; Scorretti, Riccardo; Dias, André; Dempsey, Nora M.; Bonfim, Marlio; Simonet, Pascal; Frénéa-Robin, Marie

    2017-04-01

    Magnetism and microfluidics are two key elements for the development of inexpensive and reliable tools dedicated to high-throughput biological analysis and providing a large panel of applications in domains ranging from fundamental biology to medical diagnostics. In this work, we introduce a simple protocol, relying on injection molding and reversible bonding for fabrication of magnetic cell trapping and sorting devices using only standard soft-lithography equipment. Magnetic strips or grids made of Polydimethylsiloxane (PDMS) doped with hard (NdFeB) or soft (carbonyl iron) magnetic powders were integrated at the bottom of whole PDMS chips. Preliminary results show the effective deviation/trapping of magnetic beads or magnetically-labeled bacteria as the sample flows through the microchannel, proving the potential of this rapid prototyping approach for easy fabrication of magnetic cell sorters.

  6. Microfabrication of Bubbular Cavities in PDMS for Cell Sorting and Microcell Culture Applications

    Institute of Scientific and Technical Information of China (English)

    Ut-Binh T.Giang; Michael R.King; Lisa A.DeLouise

    2008-01-01

    We describe a novel technique, low surface energy Gas Expansion Molding (GEM), to fabricate microbubble arrays in polydimethylsiloxane (PDMS) which are incorporated into parallel plate flow chambers and tested in cell sorting and microcell culture applications. This architecture confers several operational advantages that distinguish this technology approach from currently used methods. Herein we describe the GEM process and the parameters that are used to control microbubble formation and a Vacuum-Assisted Coating (VAC) process developed to selectively and spatially alter the PDMS surface chemistry in the wells and on the microchannel surface. We describe results from microflow image visualization studies conducted to investigate fluid streams above and within microbubble wells and conclude with a discussion of cell culture studies in PDMS.

  7. Approaches for cytogenetic and molecular analyses of small flow-sorted cell populations from childhood leukemia bone marrow samples

    DEFF Research Database (Denmark)

    Obro, Nina Friesgaard; Madsen, Hans Ole; Ryder, Lars Peter

    2011-01-01

    defined cell populations with subsequent analyses of leukemia-associated cytogenetic and molecular marker. The approaches described here optimize the use of the same tube of unfixed, antibody-stained BM cells for flow-sorting of small cell populations and subsequent exploratory FISH and PCR-based analyses.......Discordances between minimal residual disease estimates obtained by different methods are a problem in childhood acute lymphoblastic leukemia. We aimed to optimize methods allowing the biological exploration of such discrepancies, i.e. the combination of flow-sorting of small immunophenotypically...

  8. Widely divergent transcriptional patterns between SLE patients of different ancestral backgrounds in sorted immune cell populations.

    Science.gov (United States)

    Sharma, Shruti; Jin, Zhongbo; Rosenzweig, Elizabeth; Rao, Swapna; Ko, Kichul; Niewold, Timothy B

    2015-06-01

    Systemic lupus erythematosus (SLE) is a complex autoimmune disease of uncertain etiology. Patients from different ancestral backgrounds demonstrate differences in clinical manifestations and autoantibody profiles. We examined genome-wide transcriptional patterns in major immune cell subsets across different ancestral backgrounds. Peripheral blood was collected from African-American (AA) and European-American (EA) SLE patients and controls. CD4 T-cells, CD8 T-cells, monocytes, and B cells were purified by flow sorting, and each cell subset from each subject was run on a genome-wide expression array. Cases were compared to controls of the same ancestral background. The overlap in differentially expressed gene (DEG) lists between different cell types from the same ancestral background was modest (type between different ancestral backgrounds. IFN-stimulated gene (ISG) expression was not up-regulated synchronously in all cell types from a given patient, for example a given subject could have high ISG expression in T and B cells, but not in monocytes. AA subjects demonstrated more concordance in ISG expression between cell types from the same individual, and AA patients demonstrated significant down-regulation of metabolic gene expression which was not observed in EA patients. ISG expression was significantly decreased in B cells in patients taking immunosuppressants, while ISGs in other cell types did not differ with medication use. In conclusion, gene expression was strikingly different between immune cell subsets and between ancestral backgrounds in SLE patients. These findings emphasize the critical importance of studying multiple ancestral backgrounds and multiple cell types in gene expression studies. Ancestral backgrounds which are not studied will not benefit from personalized medicine strategies in SLE.

  9. Expression analysis of Arabidopsis vacuolar sorting receptor 3 reveals a putative function in guard cells.

    Science.gov (United States)

    Avila, Emily L; Brown, Michelle; Pan, Songqin; Desikan, Radhika; Neill, Steven J; Girke, Thomas; Surpin, Marci; Raikhel, Natasha V

    2008-01-01

    Vacuolar sorting receptors (VSRs) are responsible for the proper targeting of soluble cargo proteins to their destination compartments. The Arabidopsis genome encodes seven VSRs. In this work, the spatio-temporal expression of one of the members of this gene family, AtVSR3, was determined by RT-PCR and promoter::reporter gene fusions. AtVSR3 was expressed specifically in guard cells. Consequently, a reverse genetics approach was taken to determine the function of AtVSR3 by using RNA interference (RNAi) technology. Plants expressing little or no AtVSR3 transcript had a compressed life cycle, bolting approximately 1 week earlier and senescing up to 2 weeks earlier than the wild-type parent line. While the development and distribution of stomata in AtVSR3 RNAi plants appeared normal, stomatal function was altered. The guard cells of mutant plants did not close in response to abscisic acid treatment, and the mean leaf temperatures of the RNAi plants were on average 0.8 degrees C lower than both wild type and another vacuolar sorting receptor mutant, atvsr1-1. Furthermore, the loss of AtVSR3 protein caused the accumulation of nitric oxide and hydrogen peroxide, signalling molecules implicated in the regulation of stomatal opening and closing. Finally, proteomics and western blot analyses of cellular proteins isolated from wild-type and AtVSR3 RNAi leaves showed that phospholipase Dgamma, which may play a role in abscisic acid signalling, accumulated to higher levels in AtVSR3 RNAi guard cells. Thus, AtVSR3 may play an important role in responses to plant stress.

  10. An on-chip imaging droplet-sorting system: a real-time shape recognition method to screen target cells in droplets with single cell resolution

    Science.gov (United States)

    Girault, Mathias; Kim, Hyonchol; Arakawa, Hisayuki; Matsuura, Kenji; Odaka, Masao; Hattori, Akihiro; Terazono, Hideyuki; Yasuda, Kenji

    2017-01-01

    A microfluidic on-chip imaging cell sorter has several advantages over conventional cell sorting methods, especially to identify cells with complex morphologies such as clusters. One of the remaining problems is how to efficiently discriminate targets at the species level without labelling. Hence, we developed a label-free microfluidic droplet-sorting system based on image recognition of cells in droplets. To test the applicability of this method, a mixture of two plankton species with different morphologies (Dunaliella tertiolecta and Phaeodactylum tricornutum) were successfully identified and discriminated at a rate of 10 Hz. We also examined the ability to detect the number of objects encapsulated in a droplet. Single cell droplets sorted into collection channels showed 91 ± 4.5% and 90 ± 3.8% accuracy for D. tertiolecta and P. tricornutum, respectively. Because we used image recognition to confirm single cell droplets, we achieved highly accurate single cell sorting. The results indicate that the integrated method of droplet imaging cell sorting can provide a complementary sorting approach capable of isolating single target cells from a mixture of cells with high accuracy without any staining.

  11. Karyotyping human and mouse cells using probes from single-sorted chromosomes and open source software.

    Science.gov (United States)

    Potapova, Tamara A; Unruh, Jay R; Box, Andrew C; Bradford, William D; Seidel, Christopher W; Slaughter, Brian D; Sivagnanam, Shamilene; Wu, Yuping; Li, Rong

    2015-12-01

    Multispectral karyotyping analyzes all chromosomes in a single cell by labeling them with chromosome-specific probes conjugated to unique combinations of fluorophores. Currently available multispectral karyotyping systems require the purchase of specialized equipment and reagents. However, conventional laser scanning confocal microscopes that are capable of separating multiple overlapping emission spectra through spectral imaging and linear unmixing can be utilized for classifying chromosomes painted with multicolor probes. Here, we generated multicolor chromosome paints from single-sorted human and mouse chromosomes and developed the Karyotype Identification via Spectral Separation (KISS) analysis package, a set of freely available open source ImageJ tools for spectral unmixing and karyotyping. Chromosome spreads painted with our multispectral probe sets can be imaged on widely available spectral laser scanning confocal microscopes and analyzed using our ImageJ tools. Together, our probes and software enable academic labs with access to a laser-scanning spectral microscope to perform multicolor karyotyping in a cost-effective manner.

  12. Sorting choanoflagellates

    Science.gov (United States)

    Marconi, Veronica I.; Miño, Gaston L.; Sparacino, Javier; Banchio, Adolfo J.; Condat, Carlos A.; Koehl, Mimi A. R.; King, Nicole; Stocker, Roman

    2015-03-01

    In freshwater environments, as well as in oceans, environmental conditions are in constant fluctuation. Some heterotrophic plankton must adapt their swimming behavior in order to survive under these conditions. In the case of the choanoflagellate, the closest animal ancestor, the ability to forage for food is given not only by its single flagellum, but also by its differentiation between fast and slow swimmers. The understanding of how these cells with different strategies to swim search for food can give us a better insight into how eukaryotes respond to different stimuli. In this work, we have designed a microfluidic device that sorts choanoflagellates by their speed. The optimal geometry was found by a numerical model using the experimentally determined motilities of each swimmer type.

  13. Combining magnetic sorting of mother cells and fluctuation tests to analyze genome instability during mitotic cell aging in Saccharomyces cerevisiae.

    Science.gov (United States)

    Patterson, Melissa N; Maxwell, Patrick H

    2014-10-16

    Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on

  14. Flow sorting and exome sequencing reveal the oncogenome of primary Hodgkin and Reed-Sternberg cells.

    Science.gov (United States)

    Reichel, Jonathan; Chadburn, Amy; Rubinstein, Paul G; Giulino-Roth, Lisa; Tam, Wayne; Liu, Yifang; Gaiolla, Rafael; Eng, Kenneth; Brody, Joshua; Inghirami, Giorgio; Carlo-Stella, Carmelo; Santoro, Armando; Rahal, Daoud; Totonchy, Jennifer; Elemento, Olivier; Cesarman, Ethel; Roshal, Mikhail

    2015-02-12

    Classical Hodgkin lymphoma (cHL) is characterized by sparsely distributed Hodgkin and Reed-Sternberg (HRS) cells amid reactive host background, complicating the acquisition of neoplastic DNA without extensive background contamination. We overcame this limitation by using flow-sorted HRS and intratumor T cells and optimized low-input exome sequencing of 10 patient samples to reveal alterations in genes involved in antigen presentation, chromosome integrity, transcriptional regulation, and ubiquitination. β-2-microglobulin (B2M) is the most commonly altered gene in HRS cells, with 7 of 10 cases having inactivating mutations that lead to loss of major histocompatibility complex class I (MHC-I) expression. Enforced wild-type B2M expression in a cHL cell line restored MHC-I expression. In an extended cohort of 145 patients, the absence of B2M protein in the HRS cells was associated with lower stage of disease, younger age at diagnosis, and better overall and progression-free survival. B2M-deficient cases encompassed most of the nodular sclerosis subtype cases and only a minority of mixed cellularity cases, suggesting that B2M deficiency determines the tumor microenvironment and may define a major subset of cHL that has more uniform clinical and morphologic features. In addition, we report previously unknown genetic alterations that may render selected patients sensitive to specific targeted therapies. © 2015 by The American Society of Hematology.

  15. SorLA Controls Neurotrophic Activity by Sorting of GDNF and Its Receptors GFRα1 and RET

    Directory of Open Access Journals (Sweden)

    Simon Glerup

    2013-01-01

    Full Text Available Glial cell-line-derived neurotrophic factor (GDNF is a potent neurotrophic factor that has reached clinical trials for Parkinson’s disease. GDNF binds to its coreceptor GFRα1 and signals through the transmembrane receptor tyrosine kinase RET, or RET independently through NCAM or syndecan-3. Whereas the GDNF signaling cascades are well described, cellular turnover and trafficking of GDNF and its receptors remain poorly characterized. Here, we find that SorLA acts as sorting receptor for the GDNF/GFRα1 complex, directing it from the cell surface to endosomes. Through this mechanism, GDNF is targeted to lysosomes and degraded while GFRα1 recycles, creating an efficient GDNF clearance pathway. The SorLA/GFRα1 complex further targets RET for endocytosis but not for degradation, affecting GDNF-induced neurotrophic activities. SorLA-deficient mice display elevated GDNF levels, altered dopaminergic function, marked hyperactivity, and reduced anxiety, all of which are phenotypes related to abnormal GDNF activity. Taken together, these findings establish SorLA as a critical regulator of GDNF activity in the CNS.

  16. Real-time multivariate statistical classification of cells for flow cytometry and cell sorting: a data mining application for stem cell isolation and tumor purging

    Science.gov (United States)

    Leary, James F.; McLaughlin, Scott R.; Reece, Lisa M.; Rosenblatt, Judah I.; Hokanson, James A.

    1999-06-01

    Multivariate statistics can be used for visualization of cell subpopulations in multidimensional data space and for classification of cells within that data space. New data mining techniques we have developed, such as subtractive clustering, can be used to find the differences between test and control multiparameter flow cytometric data, e.g. in the problem of human stem cell isolation with tumor purging. They also can provide training data for subsequent multivariate statistical classification techniques such as discriminant function or logistic regression analyses. Using lookup tables, these multivariate statistical calculations can be performed in real-time, and can even include probabilities of misclassification. Thus, the only distinction between off-line classification of cells in data analysis and real-time statistical decision-making for cell sorting is the time limit in which a classification decision must be made. For real-time cell sorting we presently are able to perform these classifications in less than 625 microseconds, corresponding to the time that it takes the cell to travel from the laser intersection point to the sort decision point in a flow cytometer/cell sorter. Statistical decision making and the ability to include the costs of misclassification into that decision process will become important as flow cytometry/cell sorting moves from diagnostics to therapeutics.

  17. Automated Chemotactic Sorting and Single-cell Cultivation of Microbes using Droplet Microfluidics

    Science.gov (United States)

    Dong, Libing; Chen, Dong-Wei; Liu, Shuang-Jiang; Du, Wenbin

    2016-04-01

    We report a microfluidic device for automated sorting and cultivation of chemotactic microbes from pure cultures or mixtures. The device consists of two parts: in the first part, a concentration gradient of the chemoeffector was built across the channel for inducing chemotaxis of motile cells; in the second part, chemotactic cells from the sample were separated, and mixed with culture media to form nanoliter droplets for encapsulation, cultivation, enumeration, and recovery of single cells. Chemotactic responses were assessed by imaging and statistical analysis of droplets based on Poisson distribution. An automated procedure was developed for rapid enumeration of droplets with cell growth, following with scale-up cultivation on agar plates. The performance of the device was evaluated by the chemotaxis assays of Escherichia coli (E. coli) RP437 and E. coli RP1616. Moreover, enrichment and isolation of non-labelled Comamonas testosteroni CNB-1 from its 1:10 mixture with E. coli RP437 was demonstrated. The enrichment factor reached 36.7 for CNB-1, based on its distinctive chemotaxis toward 4-hydroxybenzoic acid. We believe that this device can be widely used in chemotaxis studies without necessarily relying on fluorescent labelling, and isolation of functional microbial species from various environments.

  18. The use of multiparameter flow cytometry and cell sorting to characterize native human bone marrow mesenchymal stem cells (MSC).

    Science.gov (United States)

    Boxall, Sally; Jones, Elena

    2015-01-01

    This chapter describes a method for identification, phenotypic analysis, and cell sorting of rare mesenchymal stem cells (MSCs) from human bone marrow (BM) aspirates. The native BM MSC population is identified based on the CD45(-/low)CD271(+) phenotype. The method consists of three related procedures: Procedure 1 involves a microbead-based pre-enrichment step. Two other procedures describe direct flow cytometric analysis of MSCs following the isolation of the mononuclear cell (MNC) fraction (Procedure 2) or more rapidly, following a simple ammonium chloride-based red cell lysis (Procedure 3). Recently described multi-lineage transcript expression in the CD45(-/low)CD271(+) cells suggests that the native BM MSC fraction could be further subdivided into functionally distinct subpopulations. The present protocols are hoped to help MSC biologists to enter this exciting field of research and to take it forward towards a better understanding of MSC biology in vivo.

  19. Detection and isolation of rare cells by 2-step enrichment high-speed flow cytometry/cell sorting and single cell LEAP laser ablation

    Science.gov (United States)

    Zordan, M. D.; Leary, James F.

    2011-02-01

    The clonal isolation of rare cells, especially cancer and stem cells, in a population is important to the development of improved medical treatment. We have demonstrated that the Laser-Enabled Analysis and Processing (LEAP, Cyntellect Inc., San Diego, CA) instrument can be used to efficiently produce single cell clones by photoablative dilution. Additionally, we have also shown that cells present at low frequencies can be cloned by photoablative dilution after they are pre-enriched by flow cytometry based cell sorting. Circulating tumor cells were modeled by spiking isolated peripheral blood cells with cells from the lung carcinoma cell line A549. Flow cytometry based cell sorting was used to perform an enrichment sort of A549 cells directly into a 384 well plate. Photoablative dilution was performed with the LEAPTM instrument to remove any contaminating cells, and clonally isolate 1 side population cell per well. We were able to isolate and grow single clones of side population cells using this method at greater than 90% efficiency. We have developed a 2 step method that is able to perform the clonal isolation of rare cells based on a medically relevant functional phenotype.

  20. Micro Flow Cytometer Chip Integrated with Micro-Pumps/Micro-Valves for Multi-Wavelength Cell Counting and Sorting

    Science.gov (United States)

    Chang, Chen-Min; Hsiung, Suz-Kai; Lee, Gwo-Bin

    2007-05-01

    Flow cytometry is a popular technique for counting and sorting of individual cells. This study presents a new chip-based flow cytometer capable of cell injection, counting and switching in an automatic format. The new microfluidic system is also capable of multi-wavelength detection of fluorescence-labeled cells by integrating multiple buried optical fibers within the chip. Instead of using large-scale syringe pumps, this study integrates micro-pumps and micro-valves to automate the entire cell injection and sorting process. By using pneumatic serpentine-shape (S-shape) micro-pumps to drive sample and sheath flows, the developed chip can generate hydrodynamic focusing to allow cells to pass detection regions in sequence. Two pairs of optical fibers are buried and aligned with the microchannels, which can transmit laser light sources with different wavelengths and can collect induced fluorescence signals. The cells labeled with different fluorescent dyes can be excited by the corresponding light source at different wavelengths. The fluorescence signals are then collected by avalanche photodiode (APD) sensors. Finally, a flow switching device composed of three pneumatic micro-valves is used for cell sorting function. Experimental data show that the developed flow cytometer can distinguish specific cells with different dye-labeling from mixed cell samples in one single process. The target cell samples can be also switched into appropriate outlet channels utilizing the proposed microvalve device. The developed microfluidic system is promising for miniature cell-based biomedical applications.

  1. Test-Retest Reliability and Internal Consistency of the Activity Card Sort-Australia (18-64).

    Science.gov (United States)

    Gustafsson, Louise; Hung, Inez Hui Min; Liddle, Jacki

    2017-01-01

    The Activity Card Sort (ACS) measures activity engagement levels. The Activity Card Sort-Australian version for adults aged 18 to 64 (ACS-Aus (18-64)) was recently developed, and psychometric properties have not yet been determined. This study was established to determine the test-retest reliability and internal consistency of the ACS-Aus (18-64) and describe activity engagement trends for healthy adults. Fifty-four adults aged 18 to 64 participated in this descriptive study. The ACS-Aus (18-64) demonstrated excellent test-retest reliability ( r = .92, p maintenance activities ( t = -2.22, p = .03), and recreation and relaxation activities ( t = -2.38, p = .02). The ACS-Aus (18-64) may be used to explore the activity engagement patterns of community-dwelling Australian adults aged 18 to 64. Further research will determine validity for clinical populations.

  2. Continuous-flow sorting of microalgae cells based on lipid content by high frequency dielectrophoresis

    Directory of Open Access Journals (Sweden)

    Doug Redelman

    2016-08-01

    Full Text Available This paper presents a continuous-flow cell screening device to isolate and separate microalgae cells (Chlamydomonas reinhardtii based on lipid content using high frequency (50 MHz dielectrophoresis. This device enables screening of microalgae due to the balance between lateral DEP forces relative to hydrodynamic forces. Positive DEP force along with amplitude-modulated electric field exerted on the cells flowing over the planar interdigitated electrodes, manipulated low-lipid cell trajectories in a zigzag pattern. Theoretical modelling confirmed cell trajectories during sorting. Separation quantification and sensitivity analysis were conducted with time-course experiments and collected samples were analysed by flow cytometry. Experimental testing with nitrogen starveddw15-1 (high-lipid, HL and pgd1 mutant (low-lipid, LL strains were carried out at different time periods, and clear separation of the two populations was achieved. Experimental results demonstrated that three populations were produced during nitrogen starvation: HL, LL and low-chlorophyll (LC populations. Presence of the LC population can affect the binary separation performance. The continuous-flow micro-separator can separate 74% of the HL and 75% of the LL out of the starting sample using a 50 MHz, 30 voltages peak-to-peak AC electric field at Day 6 of the nitrogen starvation. The separation occurred between LL (low-lipid: 86.1% at Outlet # 1 and LC (88.8% at Outlet # 2 at Day 9 of the nitrogen starvation. This device can be used for onsite monitoring; therefore, it has the potential to reduce biofuel production costs

  3. The sorting receptor Rer1 controls Purkinje cell function via voltage gated sodium channels

    Science.gov (United States)

    Valkova, Christina; Liebmann, Lutz; Krämer, Andreas; Hübner, Christian A.; Kaether, Christoph

    2017-01-01

    Rer1 is a sorting receptor in the early secretory pathway that controls the assembly and the cell surface transport of selected multimeric membrane protein complexes. Mice with a Purkinje cell (PC) specific deletion of Rer1 showed normal polarization and differentiation of PCs and normal development of the cerebellum. However, PC-specific loss of Rer1 led to age-dependent motor deficits in beam walk, ladder climbing and gait. Analysis of brain sections revealed a specific degeneration of PCs in the anterior cerebellar lobe in old animals. Electrophysiological recordings demonstrated severe deficits in spontaneous action potential generation. Measurements of resurgent currents indicated decreased surface densities of voltage-gated sodium channels (Nav), but not changes in individual channels. Analysis of mice with a whole brain Rer1-deletion demonstrated a strong down-regulation of Nav1.6 and 1.1 in the absence of Rer1, whereas protein levels of the related Cav2.1 and of Kv3.3 and 7.2 channels were not affected. The data suggest that Rer1 controls the assembly and transport of Nav1.1 and 1.6, the principal sodium channels responsible for recurrent firing, in PCs. PMID:28117367

  4. Endoplasmic reticulum membrane-sorting protein of lymphocytes (BAP31) is highly expressed in neurons and discrete endocrine cells.

    Science.gov (United States)

    Manley, H A; Lennon, V A

    2001-10-01

    BAP31 is a transmembrane protein that associates with nascent membrane proteins in transit between endoplasmic reticulum (ER) and cis-Golgi. Its C-terminal dilysine (KKEE) motif, mediating return to the ER, is consistent with a role in early sorting of membrane proteins. An initiator caspase-binding site in the C-terminal domain of BAP31 is implicated in cytoplasmic membrane fragmentation events of apoptosis. Although BAP31 RNA is ubiquitous, the protein's anatomic localization has not been determined. To gain further insight into its possible functions, we localized BAP31 in primate tissues using monoclonal antibodies. Immunoreactivity was prominent in T- and B-lymphocytes in blood and in thymus, in cerebellar Purkinje neuron bodies and dendrites, in gonadotrophs of the anterior pituitary, ovarian thecal and follicular cells, active but not quiescent thyroid epithelium, adrenal cortex more than medulla, and proximal more than distal renal tubules. Blood vessels and skeletal muscle were nonreactive. The anatomic distribution of BAP31 and the nature of proteins identified thus far as its cargo exiting the ER, suggest an interaction with proteins assembling in macromolecular complexes en route to selected sites of exocytotic and signaling activities. Apoptotic associations in mature tissues could be physiological (lymphocytes, endocrine cells) or pathological (Purkinje neurons, renal tubules).

  5. Isolation of αL I domain mutants mediating firm cell adhesion using a novel flow-based sorting method.

    Science.gov (United States)

    Pepper, Lauren R; Parthasarathy, Ranganath; Robbins, Gregory P; Dang, Nicholas N; Hammer, Daniel A; Boder, Eric T

    2013-08-01

    The inserted (I) domain of αLβ2 integrin (LFA-1) contains the entire binding site of the molecule. It mediates both rolling and firm adhesion of leukocytes at sites of inflammation depending on the activation state of the integrin. The affinity change of the entire integrin can be mimicked by the I domain alone through mutations that affect the conformation of the molecule. High-affinity mutants of the I domain have been discovered previously using both rational design and directed evolution. We have found that binding affinity fails to dictate the behavior of I domain adhesion under shear flow. In order to better understand I domain adhesion, we have developed a novel panning method to separate yeast expressing a library of I domain variants on the surface by adhesion under flow. Using conditions analogous to those experienced by cells interacting with the post-capillary vascular endothelium, we have identified mutations supporting firm adhesion that are not found using typical directed evolution techniques that select for tight binding to soluble ligands. Mutants isolated using this method do not cluster with those found by sorting with soluble ligand. Furthermore, these mutants mediate shear-driven cell rolling dynamics decorrelated from binding affinity, as previously observed for I domains bearing engineered disulfide bridges to stabilize activated conformational states. Characterization of these mutants supports a greater understanding of the structure-function relationship of the αL I domain, and of the relationship between applied force and bioadhesion in a broader context.

  6. BECN1/Beclin 1 sorts cell-surface APP/amyloid β precursor protein for lysosomal degradation.

    Science.gov (United States)

    Swaminathan, Gayathri; Zhu, Wan; Plowey, Edward D

    2016-12-01

    The regulation of plasma membrane (PM)-localized transmembrane protein/receptor trafficking has critical implications for cell signaling, metabolism and survival. In this study, we investigated the role of BECN1 (Beclin 1) in the degradative trafficking of PM-associated APP (amyloid β precursor protein), whose metabolism to amyloid-β, an essential event in Alzheimer disease, is dependent on divergent PM trafficking pathways. We report a novel interaction between PM-associated APP and BECN1 that recruits macroautophagy/endosomal regulatory proteins PIK3C3 and UVRAG. We found that BECN1 promotes surface APP internalization and sorting predominantly to endosomes and endolysosomes. BECN1 also promotes the targeting of a smaller fraction of internalized APP to LC3-positive phagophores, suggesting a role for BECN1-dependent PM macroautophagy in APP degradation. Furthermore, BECN1 facilitates lysosomal degradation of surface APP and reduces the secretion of APP metabolites (soluble ectodomains, sAPP). The association between APP and BECN1 is dependent on the evolutionarily conserved domain (ECD) of BECN1 (amino acids 267-337). Deletion of a BECN1 ECD subregion (amino acids 285-299) did not impair BECN1- PIK3C3 interaction, PtdIns3K function or macroautophagy, but was sufficient to impair the APP-BECN1 interaction and BECN1's effects on surface APP internalization and degradation, resulting in increased secretion of sAPPs. Interestingly, both the BECN1-APP association and BECN1-dependent APP endocytosis and degradative trafficking were negatively regulated by active AKT. Our results further implicate phosphorylation of the BECN1 Ser295 residue in the inhibition of APP degradation by AKT. Our studies reveal a novel function for BECN1 in the sorting of a plasma membrane protein for endolysosomal and macroautophagic degradation.

  7. Identification of nitrite-reducing bacteria using sequential mRNA fluorescence in situ hybridization and fluorescence-assisted cell sorting.

    Science.gov (United States)

    Mota, Cesar R; So, Mark Jason; de los Reyes, Francis L

    2012-07-01

    Sequential mRNA fluorescence in situ hybridization (mRNA FISH) and fluorescence-assisted cell sorting (SmRFF) was used for the identification of nitrite-reducing bacteria in mixed microbial communities. An oligonucleotide probe labeled with horseradish peroxidase (HRP) was used to target mRNA of nirS, the gene that encodes nitrite reductase, the enzyme responsible for the dissimilatory reduction of nitrite to nitric oxide. Clones for nirS expression were constructed and used to provide proof of concept for the SmRFF method. In addition, cells from pure cultures of Pseudomonas stutzeri and denitrifying activated sludge were hybridized with the HRP probe, and tyramide signal amplification was performed, conferring a strongly fluorescent signal to cells containing nirS mRNA. Flow cytometry-assisted cell sorting was used to detect and physically separate two subgroups from a mixed microbial community: non-fluorescent cells and an enrichment of fluorescent, nitrite-reducing cells. Denaturing gradient gel electrophoresis (DGGE) and subsequent sequencing of 16S ribosomal RNA (rRNA) genes were used to compare the fragments amplified from the two sorted subgroups. Sequences from bands isolated from DGGE profiles suggested that the dominant, active nitrite reducers were closely related to Acidovorax BSB421. Furthermore, following mRNA FISH detection of nitrite-reducing bacteria, 16S rRNA FISH was used to detect ammonia-oxidizing and nitrite-oxidizing bacteria on the same activated sludge sample. We believe that the molecular approach described can be useful as a tool to help address the longstanding challenge of linking function to identity in natural and engineered habitats.

  8. Sorting methods of breast cancer stem cells%乳腺肿瘤干细胞分选的相关研究

    Institute of Scientific and Technical Information of China (English)

    郭崇勇; 宋科瑛; 李克

    2011-01-01

    In recent years, the theory of cancer stem cells has provided a new perspective on the treatment of cancers including breast cancer. The accurate sorting of breast cancer stem cells is critical. The sorting procedure is consist of 4 steps: isolation of side population, serum-free suspension culture,determination of specific cell surface markers and of the activity of aldehyde dehydrogenase 1 ( ALDH1 ) enzymatic through the ALDEFLUOR assay. Some studies choose drug resistance as an additional method for sorting cancer stem cells because of the enrichment of cancer stem cells after chemotherapy . The controversy about the sorting outcome of breast cancer stem cell mainly focus on molecular markers like CD44+ CD24- and ALDH1+ . The problem needed to be settled is to identify which sorting method and markers are appropriate.%"肿瘤干细胞"学说的提出为肿瘤包括乳腺肿瘤在内的治疗提供了新的思路.乳腺肿瘤干细胞正确的分选纯化是研究的关键,其分选主要历经侧群细胞分选、无血清悬浮培养、细胞表面分子标记分选、ALDEFLUOR试剂盒检测乙醛脱氢酶1高活性分选法4个阶段;由于化疗会富集肿瘤干细胞,部分学者把化疗耐药也作为一种分选的方法.目前有关乳腺肿瘤干细胞分选结果的争论主要集中在标志物CD44+ CD24-、ALDH1+ 之间.哪种分选方法或标志物的选择更适宜分选乳腺肿瘤干细胞成为亟待解决的问题.

  9. Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35

    Institute of Scientific and Technical Information of China (English)

    Mingmin Yan; Shanping Mao; Huimin Dong; Baohui Liu; Qian Zhang; Gaofeng Pan; Zhiping Fu

    2012-01-01

    PC12 cell injury was induced using 20 μM amyloid β-protein 25-35 to establish a model of Alzheimer's disease.The cells were then treated with 5, 10, and 25 μM Schisandrin B.Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25-35 gradually increased and the rate of apoptosis gradually decreased.Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased.Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change.These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25-35 in a dose-dependent manner.This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein.PC12 cell injury was induced using 20 μM amyloid β-protein 25-35 to establish a model of Alzheimer's disease.The cells were then treated with 5, 10, and 25 μM Schisandrin B.Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25-35 gradually increased and the rate of apoptosis gradually decreased.Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased.Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change.These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25

  10. Generation of Recombinant Monoclonal Antibodies from Immunised Mice and Rabbits via Flow Cytometry and Sorting of Antigen-Specific IgG+ Memory B Cells.

    Directory of Open Access Journals (Sweden)

    Dale O Starkie

    Full Text Available Single B cell screening strategies, which avoid both hybridoma fusion and combinatorial display, have emerged as important technologies for efficiently sampling the natural antibody repertoire of immunized animals and humans. Having access to a range of methods to interrogate different B cell subsets provides an attractive option to ensure large and diverse panels of high quality antibody are produced. The generation of multiple antibodies and having the ability to find rare B cell clones producing IgG with unique and desirable characteristics facilitates the identification of fit-for-purpose molecules that can be developed into therapeutic agents or research reagents. Here, we describe a multi-parameter flow cytometry single-cell sorting technique for the generation of antigen-specific recombinant monoclonal antibodies from single IgG+ memory B cells. Both mouse splenocytes and rabbit PBMC from immunised animals were used as a source of B cells. Reagents staining both B cells and other unwanted cell types enabled efficient identification of class-switched IgG+ memory B cells. Concurrent staining with antigen labelled separately with two spectrally-distinct fluorophores enabled antigen-specific B cells to be identified, i.e. those which bind to both antigen conjugates (double-positive. These cells were then typically sorted at one cell per well using FACS directly into a 96-well plate containing reverse transcriptase reaction mix. Following production of cDNA, PCR was performed to amplify cognate heavy and light chain variable region genes and generate transcriptionally-active PCR (TAP fragments. These linear expression cassettes were then used directly in a mammalian cell transfection to generate recombinant antibody for further testing. We were able to successfully generate antigen-specific recombinant antibodies from both the rabbit and mouse IgG+ memory B cell subset within one week. This included the generation of an anti-TNFR2 blocking

  11. Unequivocal identification of subpopulations in putative multiclonal Trypanosoma cruzi strains by FACs single cell sorting and genotyping.

    Directory of Open Access Journals (Sweden)

    Helder Magno Silva Valadares

    Full Text Available Trypanosoma cruzi, the etiological agent of Chagas disease, is a polymorphic species. Evidence suggests that the majority of the T. cruzi populations isolated from afflicted humans, reservoir animals, or vectors are multiclonal. However, the extent and the complexity of multiclonality remain to be established, since aneuploidy cannot be excluded and current conventional cloning methods cannot identify all the representative clones in an infection. To answer this question, we adapted a methodology originally described for analyzing single spermatozoids, to isolate and study single T. cruzi parasites. Accordingly, the cloning apparatus of a Fluorescence-Activated Cell Sorter (FACS was used to sort single T. cruzi cells directly into 96-wells microplates. Cells were then genotyped using two polymorphic genomic markers and four microsatellite loci. We validated this methodology by testing four T. cruzi populations: one control artificial mixture composed of two monoclonal populations--Silvio X10 cl1 (TcI and Esmeraldo cl3 (TcII--and three naturally occurring strains, one isolated from a vector (A316A R7 and two others derived from the first reported human case of Chagas disease. Using this innovative approach, we were able to successfully describe the whole complexity of these natural strains, revealing their multiclonal status. In addition, our results demonstrate that these T. cruzi populations are formed of more clones than originally expected. The method also permitted estimating of the proportion of each subpopulation of the tested strains. The single-cell genotyping approach allowed analysis of intrapopulation diversity at a level of detail not achieved previously, and may thus improve our comprehension of population structure and dynamics of T. cruzi. Finally, this methodology is capable to settle once and for all controversies on the issue of multiclonality.

  12. Lovastatin-induced cholesterol depletion affects both apical sorting and endocytosis of aquaporin-2 in renal cells.

    Science.gov (United States)

    Procino, G; Barbieri, C; Carmosino, M; Rizzo, F; Valenti, G; Svelto, M

    2010-02-01

    Vasopressin causes the redistribution of the water channel aquaporin-2 (AQP2) from cytoplasmic storage vesicles to the apical plasma membrane of collecting duct principal cells, leading to urine concentration. The molecular mechanisms regulating the selective apical sorting of AQP2 are only partially uncovered. In this work, we investigate whether AQP2 sorting/trafficking is regulated by its association with membrane rafts. In both MCD4 cells and rat kidney, AQP2 preferentially associated with Lubrol WX-insoluble membranes regardless of its presence in the storage compartment or at the apical membrane. Block-and-release experiments indicate that 1) AQP2 associates with detergent-resistant membranes early in the biosynthetic pathway; 2) strong cholesterol depletion delays the exit of AQP2 from the trans-Golgi network. Interestingly, mild cholesterol depletion promoted a dramatic accumulation of AQP2 at the apical plasma membrane in MCD4 cells in the absence of forskolin stimulation. An internalization assay showed that AQP2 endocytosis was clearly reduced under this experimental condition. Taken together, these data suggest that association with membrane rafts may regulate both AQP2 apical sorting and endocytosis.

  13. Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35.

    Science.gov (United States)

    Yan, Mingmin; Mao, Shanping; Dong, Huimin; Liu, Baohui; Zhang, Qian; Pan, Gaofeng; Fu, Zhiping

    2012-03-25

    PC12 cell injury was induced using 20 μM amyloid β-protein 25-35 to establish a model of Alzheimer's disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25-35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased. Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change. These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25-35 in a dose-dependent manner. This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein.

  14. Sorting of Streptomyces cell pellets using a complex object parametric analyzer and sorter

    NARCIS (Netherlands)

    Petrus, Marloes L C; van Veluw, G Jerre; Wösten, Han A B; Claessen, Dennis; Wosten, Han

    2014-01-01

    Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these organisms form networks of interconnected hyphae, known as pellets, which are heterogeneous in size. Here we describe a method to analyze and sort my

  15. Sorting of Streptomyces cell pellets using a complex object parametric analyzer and sorter

    NARCIS (Netherlands)

    Petrus, Marloes L C; van Veluw, G Jerre; Wösten, Han A B; Claessen, Dennis; Wosten, Han

    2014-01-01

    Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these organisms form networks of interconnected hyphae, known as pellets, which are heterogeneous in size. Here we describe a method to analyze and sort

  16. Cell sorting enables interphase fluorescence in situ hybridization detection of low BCR-ABL1 producing stem cells in chronic myeloid leukaemia patients beyond deep molecular remission.

    Science.gov (United States)

    van Kooten Niekerk, Peter B; Petersen, Charlotte C; Nyvold, Charlotte G; Ommen, Hans B; Roug, Anne S; Nederby, Line; Hokland, Peter; Kjeldsen, Eigil

    2014-01-01

    The exact disease state of chronic myeloid leukaemia (CML) patients in deep molecular remission is unknown, because even the most sensitive quantitative reverse transcription polymerase chain reaction (qPCR) methods cannot identify patients prone to relapse after treatment withdrawal. To elucidate this, CD34(+) stem cell and progenitor cell subpopulations were isolated by fluorescence-activated cell sorting (FACS), and their content of residual Philadelphia positive (Ph(+) ) cells was evaluated in 17 CML patients (major molecular response, n = 6; 4-log reduction in BCR-ABL1 expression (MR(4) ), n = 11) using both sensitive qPCR and interphase fluorescence in situ hybridization (iFISH). Despite evaluating fewer cells, iFISH proved superior to mRNA-based qPCR in detecting residual Ph(+) stem cells (P = 0·005), and detected Ph(+) stem- and progenitor cells in 9/10 patients at frequencies of 2-14%. Moreover, while all qPCR(+) samples also were iFISH(+) , 9/33 samples were qPCR-/iFISH(+) , including all positive samples from MR(4) patients. Our findings show that residual Ph(+) cells are low BCR-ABL1 producers, and that DNA-based methods are required to assess the content of persisting Ph(+) stem cells in these patients. This approach demonstrates a clinically applicable manner of assessing residual disease at the stem cell level in CML patients in MR(4) , and may enable early and safe identification of candidates for tyrosine kinase inhibitor withdrawal.

  17. Immunoglobulin characteristics and RNAseq data of FcRL4+ B cells sorted from synovial fluid and tissue of patients with rheumatoid arthritis.

    Science.gov (United States)

    Amara, Khaled; Clay, Elizabeth; Yeo, Lorraine; Ramsköld, Daniel; Spengler, Julia; Sippl, Natalie; Cameron, James; Israelsson, Lena; Titcombe, Philip J; Grönwall, Caroline; Sahbudin, Ilfita; Filer, Andrew; Raza, Karim; Malmström, Vivianne; Scheel-Toellner, Dagmar

    2017-08-01

    This manuscript is a companion paper to Amara et al. [1]. Data shown here include detailed clinical characteristics from anonymized patients, the Ig subclass data generated from B cells sorted from four individual patients, tables detailing variable gene region sequences from sorted cells linked to the patient information and the sequence yields from individual patients. Furthermore a URL link to the RNAseq datasets submitted to GEO is included.

  18. DNA-encoded antibody libraries: a unified platform for multiplexed cell sorting and detection of genes and proteins.

    Science.gov (United States)

    Bailey, Ryan C; Kwong, Gabriel A; Radu, Caius G; Witte, Owen N; Heath, James R

    2007-02-21

    Whether for pathological examination or for fundamental biology studies, different classes of biomaterials and biomolecules are each measured from a different region of a typically heterogeneous tissue sample, thus introducing unavoidable sources of noise that are hard to quantitate. We describe the method of DNA-encoded antibody libraries (DEAL) for spatially multiplexed detection of ssDNAs and proteins as well as for cell sorting, all on the same diagnostic platform. DEAL is based upon the coupling of ssDNA oligomers onto antibodies which are then combined with the biological sample of interest. Spotted DNA arrays, which are found to inhibit biofouling, are utilized to spatially stratify the biomolecules or cells of interest. We demonstrate the DEAL technique for (1) the rapid detection of multiple proteins within a single microfluidic channel, and, with the additional step of electroless amplification of gold-nanoparticle labeled secondary antibodies, we establish a detection limit of 10 fM for the protein IL-2, 150 times more sensitive than the analogue ELISA; (2) the multiplexed, on-chip sorting of both immortalized cell lines and primary immune cells with an efficiency that exceeds surface-confined panning approaches; and (3) the co-detection of ssDNAs, proteins, and cell populations on the same platform.

  19. Enzymatic sorting of bacterial colonies on filter paper replicas: detection of labile activities.

    Science.gov (United States)

    Bulawa, C E; Ganong, B R; Sparrow, C P; Raetz, C R

    1981-01-01

    To utilize autoradiographic colony-sorting techniques (C. R. H. Raetz, Proc. Natl. Acad. Sci. U.S.A. 72:2274-2278, 1975) for the isolation of mutants with unstable enzymes, we report a new desiccation-induced lysis method, compatible with low temperatures. Furthermore, a general, two-step protocol is presented for clonal detection of hydrolytic reactions. The advantages of these critical modifications are demonstrated with the membrane enzymes glycerol 3-phosphate acyltransferase and cytidine 5'-diphosphate-diglyceride hydrolase. Images PMID:6116698

  20. Fluorescence in situ hybridization-flow cytometry-cell sorting-based method for separation and enrichment of type I and type II methanotroph populations.

    Science.gov (United States)

    Kalyuzhnaya, Marina G; Zabinsky, Rebecca; Bowerman, Sarah; Baker, David R; Lidstrom, Mary E; Chistoserdova, Ludmila

    2006-06-01

    A fluorescence in situ hybridization-flow cytometry (FISH/FC)-based method was optimized using artificial mixtures of pure cultures of methanotrophic bacteria. Traditional oligonucleotide probes targeting 16S rRNAs of type I (MG84/705 probe) and type II (MA450 probe) methanotrophs were labeled with fluorescein or Alexa fluor and used for FISH, followed by fluorescence-activated FC analysis and cell sorting (FACS). The method resulted in efficient separation of target cells (type I or type II methanotrophs) from the artificial mixtures. The method was then applied for detection and enrichment of type I and type II methanotroph populations from a natural sample, Lake Washington sediment. Cells were extracted from the sediment, fixed, and subjected to FISH/FC/FACS. The resulting subpopulations were analyzed by reverse transcriptase PCR surveys of 16S rRNA, pmoA (encoding a subunit of particulate methane monooxygenase), and fae (encoding formaldehyde-activating enzyme) genes. The functional gene analysis indicated specific separation of the type I and type II methanotroph populations. 16S rRNA gene analysis revealed that type I methanotrophs comprised 59% of the subpopulation separated using the type I-specific probe and that type II methanotrophs comprised 47.5% of the subpopulation separated using the type II-specific probe. Our data indicate that the FISH/FC/FACS protocol described can provide significant enrichment of microbial populations of interest from complex natural communities and that these can be used for genetic tests. We further tested the possibility of direct whole-genome amplification (WGA) from limited numbers of sorted cells, using artificial mixtures of microbes whose genome sequences are known. We demonstrated that efficient WGA can be achieved using 10(4) or more cells separated by 16S rRNA-specific FISH/FC/FACS, while fewer cells resulted in less specific WGA.

  1. A specific sorting signal is not required for the polarized secretion of newly synthesized proteins from cultured intestinal epithelial cells.

    Science.gov (United States)

    Rindler, M J; Traber, M G

    1988-08-01

    Caco-2 cells, derived from human colon, have the morphological, functional, and biochemical properties of small intestinal epithelial cells. After infection with enveloped viruses, influenza virions assembled at the apical plasma membrane while vesicular stomatitis virus (VSV) particles appeared exclusively at the basolateral membrane, similar to the pattern observed in virus-infected Madin-Darby canine kidney (MDCK). When grown in Millicell filter chamber devices and labeled with [35S]methionine, Caco-2 monolayers released all of their radiolabeled secretory products preferentially into the basal chamber. Among the proteins identified were apolipoproteins AI and E, transferrin, and alpha-fetoprotein. No proteins were observed to be secreted preferentially from the apical cell surface. The lysosomal enzyme beta-hexosaminidase was also secreted primarily from the basolateral surface of the cells in the presence or absence of lysosomotropic drugs or tunicamycin, which inhibit the targetting of lysosomal enzymes to lysosomes. Neither of these drug treatments significantly affected the polarized secretion of other nonlysosomal proteins. In addition, growth hormone (GH), which is released in a nonpolar fashion from MDCK cells, was secreted exclusively from the basolateral membrane after transfection of Caco-2 cells with GH cDNA in a pSV2-based expression vector. Similar results were obtained in transient expression experiments and after selection of permanently transformed Caco-2 cells expressing GH. Since both beta-hexosaminidase and GH would be expected to lack sorting signals for polarized exocytosis in epithelial cells, these results indicate that in intestinal cells, proteins transported via the basolateral secretory pathway need not have specific sorting signals.

  2. Combined use of immunomagnetic activated cell sorting technique enrichment and immunocytochemistry with hematoxylin and eosin staining for identification of circulating tumor cells in peripheral blood mononuclcar cells of hepatocellular carcinoma patients%应用免疫激活磁珠分选技术CD45去除方法富集——免疫细胞化学联合苏木素-伊红染色检测肝癌患者循环肿瘤细胞

    Institute of Scientific and Technical Information of China (English)

    郭立民; 鲁岩; 彭吉润; 蒋力

    2014-01-01

    Objective To estimate the applied value of magnetic activated cell sorting (MACS) techniques with CD45 depletion and immunocytochemistry in combination with hematoxylin and eosin (HE) staining in identifying circulating tumor cells (CTCs) in peripheral blood mononuclear cells (PBMC) of hepatocellular carcinoma (HCC) patients.Methods The expression of CK (CK8,CK18,and CK19) was detected in 18 epithelia-derived tumor cell lines including 9 human hepatocellular carcinoma cell lines.The peripheral blood of HCC patients and healthy volunteers was collected for determination of CTCs in PBMC from HCC patients using MACS techniques with CD45 depletion and immunocytochemstry in combination with HE staining.Results The expression rate of CK8,CK18 and CK19 in the selected CTCs was 72.22%,83.33% and 66.67% respectively.CKs were detected in most of the 9 hepatocelluar carcinoma cell lines.We found intact CTCs in PBMC from HCC patients using HE staining and immunocytochemistry after PBMC enrichment by MACS techniques with CD45 + depletion.The sensitivity of this method was up to 63.15%,and no CTCs were detected in PBMC from 20 healthy controls.Conclusion CKs could be a tumor marker for detection of CTCs in HCC patients.The method of HE staining and immunocytochemistry after PBMC enrichment by MACS technique with CD45 + depletion has potentials in detection of circulating HCC cells.%目的 探讨免疫激活磁珠分选(MACS) CD45去除方法富集后,以细胞角蛋白(CK)为标记联合苏木素-伊红(HE)染色检测肝癌患者外周血肿瘤细胞(CTC)的价值.方法 应用逆转录-聚合酶链反应(RT-PCR)检测18种上皮肿瘤细胞株的CK(CK8、CK18、CK19)表达;采集健康志愿者、肝癌患者外周血,以MACS技术CD45去除方法对外周血单个核细胞(PBMC)进行富集,以CK为标记,采用免疫组织化学染色联合HE染色检测肿瘤细胞.结果 在18种上皮性肿瘤细胞株中CK8、CK18、CK19表达率分别为72.22%、83.33%和66

  3. Design of a multi-stage microfluidics system for high-speed flow cytometry and closed system cell sorting for cytomics

    Science.gov (United States)

    Grafton, Meggie; Reece, Lisa M.; Irazoqui, Pedro P.; Jung, Byunghoo; Summers, Huw D.; Bashir, Rashid; Leary, James F.

    2008-02-01

    To produce a large increase in total throughput, a multi-stage microfluidics system (US Patent pending) is being developed for flow cytometry and closed system cell sorting. The multi-stage system provides for sorting and re-sorting of cohorts of cells beginning with multiple cells per sorting unit in the initial stages of the microfluidic device and achieving single cell sorting at subsequent stages. This design theoretically promises increases of 2- or 3-orders of magnitude in total cell throughput needed for cytomics applications involving gene chip or proteomics analyses of sorted cell subpopulations. Briefly, silicon wafers and CAD software were used with SU-8 soft photolithography techniques and used as a mold to create Y-shaped, multi-stage microfluidic PDMS chips. PDMS microfluidic chips were fabricated and tested using fluorescent microspheres driven through the chip by a microprocessor-controlled syringe drive and excited on an inverted Nikon fluorescence microscope. Inter-particle spacings were measured and used as experimental data for queuing theory models of multi-stage system performance. A miniaturized electronics system is being developed for a small portable instrument. A variety of LED light sources, waveguides, and APD detectors are being tested to find optimal combinations for creating an LED-APD configuration at the entry points of the Y-junctions for the multi-stage optical PDMS microfluidic chips. The LEDs, APDs, and PDMS chips are being combined into an inexpensive, small portable, closed system sorter suitable for operation inside a standard biohazard hood for both sterility and closed system cell sorting as an alternative to large, expensive, and conventional droplet-based cell sorters.

  4. Diurnal activity of soil surface arthropods in agroecosystems: design for an inexpensive time-sorting pitfall trap

    Energy Technology Data Exchange (ETDEWEB)

    Blumberg, A.Y.; Crossley, D.A. Jr.

    1986-01-01

    The design for an inexpensive time-sorting pitfall trap is presented. The basis of the mechanism is a rotary stepping solenoid powered by lantern batteries. Traps were utilized to sample soil surface arthropods at two hour intervals for five 24 hr periods in 1983. One trap each was placed in conventional tillage (CT) and no-tillage (NT) agroecosystems. Soil arthropod surface activity was greatest in CT on 9 July during the dawn and dusk periods but the data did not indicate other dominant trends. Activity in NT was greatest during dusk on 27 June, but again no other dominant trends were evident. When CT and NT are combined over the sample dates, surface soil arthropod activity peaked during dusk, with a smaller activity peak at dawn. 10 refs., 6 figs.

  5. Dynamic GLUT4 sorting through a syntaxin-6 compartment in muscle cells is derailed by insulin resistance-causing ceramide.

    Science.gov (United States)

    Foley, Kevin P; Klip, Amira

    2014-04-04

    GLUT4 constitutively recycles between the plasma membrane and intracellular depots. Insulin shifts this dynamic equilibrium towards the plasma membrane by recruiting GLUT4 to the plasma membrane from insulin-responsive vesicles. Muscle is the primary site for dietary glucose deposition; however, how GLUT4 sorts into insulin-responsive vesicles, and if and how insulin resistance affects this process, is unknown. In L6 myoblasts stably expressing myc-tagged GLUT4, we analyzed the intracellular itinerary of GLUT4 as it internalizes from the cell surface and examined if such sorting is perturbed by C2-ceramide, a lipid metabolite causing insulin resistance. Surface-labeled GLUT4myc that internalized for 30 min accumulated in a Syntaxin-6 (Stx6)- and Stx16-positive perinuclear sub-compartment devoid of furin or internalized transferrin, and displayed insulin-responsive re-exocytosis. C2-ceramide dispersed the Stx6-positive sub-compartment and prevented insulin-responsive re-exocytosis of internalized GLUT4myc, even under conditions not affecting insulin-stimulated signaling towards Akt. Microtubule disruption with nocodazole prevented pre-internalized GLUT4myc from reaching the Stx6-positive perinuclear sub-compartment and from undergoing insulin-responsive exocytosis. Removing nocodazole allowed both parameters to recover, suggesting that the Stx6-positive perinuclear sub-compartment was required for GLUT4 insulin-responsiveness. Accordingly, Stx6 knockdown inhibited by ∼50% the ability of internalized GLUT4myc to undergo insulin-responsive re-exocytosis without altering its overall perinuclear accumulation. We propose that Stx6 defines the insulin-responsive compartment in muscle cells. Our data are consistent with a model where ceramide could cause insulin resistance by altering intracellular GLUT4 sorting.

  6. Flow virometric sorting and analysis of HIV quasispecies from plasma

    Science.gov (United States)

    Jones, Jennifer C.; Keele, Brandon F.; Jenkins, Lisa M. Miller; Demberg, Thorsten

    2017-01-01

    Flow cytometry is utilized extensively for cellular analysis, but technical limitations have prevented its routine application for characterizing virus. The recent introduction of nanoscale fluorescence-activated cytometric cell sorting now allows analysis of individual virions. Here, we demonstrate staining and sorting of infectious HIV. Fluorescent antibodies specific for cellular molecules found on budding virions were used to label CCR5-tropic Bal HIV and CXCR4-tropic NL4.3 HIV Env-expressing pseudovirions made in THP-1 cells (monocyte/macrophage) and H9 cells (T cells), respectively. Using a flow cytometer, we resolved the stained virus beyond isotype staining and demonstrated purity and infectivity of sorted virus populations on cells with the appropriate coreceptors. We subsequently sorted infectious simian/human immunodeficiency virus from archived plasma. Recovery was approximately 0.5%, but virus present in plasma was already bound to viral-specific IgG generated in vivo, likely contributing to the low yield. Importantly, using two broadly neutralizing HIV antibodies, PG9 and VRC01, we also sorted virus from archived human plasma and analyzed the sorted populations genetically and by proteomics, identifying the quasispecies present. The ability to sort infectious HIV from clinically relevant samples provides material for detailed molecular, genetic, and proteomic analyses applicable to future design of vaccine antigens and potential development of personalized treatment regimens. PMID:28239654

  7. Resolving sorting mechanisms into exosomes

    NARCIS (Netherlands)

    Stoorvogel, Willem

    2015-01-01

    The complexity of mechanisms driving protein sorting into exosomes is only beginning to emerge. In a paper recently published in Cell Research, Roucourt et al. report that trimming of heparan sulfate side chains of syndecans by endosomal heparanase facilitates sorting into exosomes by the formation

  8. Quantitative and qualitative analysis of small RNAs in human endothelial cells and exosomes provides insights into localized RNA processing, degradation and sorting

    NARCIS (Netherlands)

    van Balkom, Bas W M; Eisele, Almut S; Pegtel, D Michiel; Bervoets, Sander; Verhaar, Marianne C

    2015-01-01

    Exosomes are small vesicles that mediate cell-cell communication. They contain proteins, lipids and RNA, and evidence is accumulating that these molecules are specifically sorted for release via exosomes. We recently showed that endothelial-cell-produced exosomes promote angiogenesis in vivo in a sm

  9. Application of flow cytometry and cell sorting to the bacterial analysis of environmental aerosol samples.

    Science.gov (United States)

    Hernlem, Bradley J; Ravva, Subbarao V

    2007-12-01

    Flow cytometry (FCM) combined with viability staining is a useful tool in discerning viable bacteria in environmental samples where traditional culture methods may fail. Contamination of aerosol samples with dust and other non-biological particles can interfere with accurate sample analysis and therefore there is a desire to exclude those particles from analysis. Particles were sorted according to their light scattering properties, cultured and isolates obtained. Isolates were cultured in suspension and reanalyzed by flow cytometry. The isolates were also analyzed and identified by DNA sequence analysis. Isolates with statistically similar light scattering properties shared common sequence identification. Isolates exhibited distinct light scattering profiles that roughly correlated with their originating gate, but often the peak of the profile was outside that gate.

  10. 3种花色苷对DF-1细胞的影响%Effect of Three Sorts of Anthocyanin on DF-1 Cells

    Institute of Scientific and Technical Information of China (English)

    盖丽丽; 张莉; 姜世金; 雷用东

    2012-01-01

    Objective: To investigate the impact of three sorts of anthocyanin including heart radish anthocyanin, purple potato anthocyanin and purple corn anthocyanin on DF-1 cell line. Methods: The impact of the three sorts of anthocyanin on DF-1 cells growth was explored and the final concentration was measured respectively by intuitive observation. The impact of the three sorts of anthocyanin in improving the role of cell activity was explored by MTT assay. Results: The different concentrations of anthocyanins were affected on DF-1 monolayer cells growth, and the optimal final concentration of heart radish anthocyanin, purple potato anthocyanin and purple corn anthocyanin was got for 100, 75 and 50 μg/mL respectively. At the same time, we mapped the chart based on the data measured by MTT assay. Conclusion: Different varieties of anthocyanins as different concentrations were affected on DF-1 monolayer cells growth significantly. These will provide data support for the research of anthocyanins antivirus in the future.%目的:探讨来自心里美萝卜、紫甘薯和紫玉米的3种不同的花色苷对DF-1细胞单层生长的影响.方法:直观观察3种花色苷对DF-1细胞生长的影响,初步摸索其最适终浓度;用MTT法检测花色苷在提高细胞活性方面的作用.结果:不同浓度的3种花色苷均对细胞单层生长有不同的影响,心里美萝卜花色苷、紫甘薯花色苷和紫玉米花色苷对细胞生长的最适终浓度分别为100、75和50 μg/mL;用MTT法获得相应数据并制成细胞活性图表.结论:不同品种来源及不同浓度的花色苷对DF-1细胞单层生长均有明显影响,为今后开展花色苷促细胞生长,提高细胞抗病毒活性研究提供了数据基础.

  11. Antibody-free magnetic cell sorting of genetically modified primary human CD4+ T cells by one-step streptavidin affinity purification.

    Directory of Open Access Journals (Sweden)

    Nicholas J Matheson

    Full Text Available Existing methods for phenotypic selection of genetically modified mammalian cells suffer disadvantages of time, cost and scalability and, where antibodies are used to bind exogenous cell surface markers for magnetic selection, typically yield cells coated with antibody-antigen complexes and beads. To overcome these limitations we have developed a method termed Antibody-Free Magnetic Cell Sorting in which the 38 amino acid Streptavidin Binding Peptide (SBP is displayed at the cell surface by the truncated Low Affinity Nerve Growth Receptor (LNGFRF and used as an affinity tag for one-step selection with streptavidin-conjugated magnetic beads. Cells are released through competition with the naturally occurring vitamin biotin, free of either beads or antibody-antigen complexes and ready for culture or use in downstream applications. Antibody-Free Magnetic Cell Sorting is a rapid, cost-effective, scalable method of magnetic selection applicable to either viral transduction or transient transfection of cell lines or primary cells. We have optimised the system for enrichment of primary human CD4+ T cells expressing shRNAs and exogenous genes of interest to purities of >99%, and used it to isolate cells following Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9 genome editing.

  12. Flow sorting in aquatic ecology

    Directory of Open Access Journals (Sweden)

    Marcus Reckermann

    2000-06-01

    Full Text Available Flow sorting can be a very helpful tool in revealing phytoplankton and bacterial community structure and elaborating specific physiological parameters of isolated species. Droplet sorting has been the most common technique. Despite the high optical and hydro-dynamic stress for the cells to be sorted, many species grow in culture subsequent to sorting. To date, flow sorting has been applied to post-incubation separation in natural water samples to account for group-specific physiological parameters (radiotracer-uptake rates, to the production of clonal or non-clonal cultures from mixtures, to the isolaton of cell groups from natural assemblages for molecular analyses, and for taxonomic identification of sorted cells by microscopy. The application of cell sorting from natural water samples from the Wadden Sea, including different cryptophytes, cyanobacteria and diatoms, is shown, as well as the establishment of laboratory cultures from field samples. The optional use of a red laser to account for phycocyanine-rich cells is also discussed.

  13. Nanoparticle-based sorting of circulating tumor cells by epithelial antigen expression during disease progression in an animal model.

    Science.gov (United States)

    Muhanna, Nidal; Mepham, Adam; Mohamadi, Reza M; Chan, Harley; Khan, Tahsin; Akens, Margarete; Besant, Justin D; Irish, Jonathan; Kelley, Shana O

    2015-10-01

    Circulating tumor cells (CTCs) can be used as markers for the detection, characterization, and targeted therapeutic management of cancer. We recently developed a nanoparticle-mediated approach for capture and sorting of CTCs based on their specific epithelial phenotype. In the current study, we investigate the phenotypic transition of tumor cells in an animal model and show the correlation of this transition with tumor progression. VX2 tumor cells were injected into rabbits, and CTCs were evaluated during tumor progression and correlated with computerized tomography (CT) measurements of tumor volume. The results showed a dramatic increase of CTCs during the four weeks of tumor growth. Following resection, CTC levels dropped but then rebounded, likely due to lymph node metastases. Additionally, CTCs showed a marked loss of the epithelial cell adhesion molecule (EpCAM) relative to precursor cells. In conclusion, the device accurately traces disease progression and CTC phenotypic shift in an animal model. The detection of circulating tumor cells (CTCs) has been used to predict disease prognosis. In this study, the authors developed a nanoparticle-mediated platform based on microfluidics to analyze the differential expressions of epithelial cell adhesion molecule (EpCAM) on CTCs in an animal model. It was found that the loss of EpCAM correlated with disease progression. Hence, the use of this platform may be further applied in other cancer models in the future. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Efficient Parallel Sorting for Migrating Birds Optimization When Solving Machine-Part Cell Formation Problems

    National Research Council Canada - National Science Library

    Soto, Ricardo; Crawford, Broderick; Almonacid, Boris; Paredes, Fernando

    2016-01-01

    The Machine-Part Cell Formation Problem (MPCFP) is a NP-Hard optimization problem that consists in grouping machines and parts in a set of cells, so that each cell can operate independently and the intercell movements are minimized...

  15. Slashing the timelines: Opting to generate high-titer clonal lines faster via viability-based single cell sorting.

    Science.gov (United States)

    Misaghi, Shahram; Shaw, David; Louie, Salina; Nava, Adrian; Simmons, Laura; Snedecor, Brad; Poon, Chungkee; Paw, Jonathan S; Gilmour-Appling, Laurie; Cupp, James E

    2016-01-01

    Chinese hamster ovary (CHO) cell line development (CLD) is a long and laborious process, which requires up to 5 - 6 months in order to generate and bank CHO lines capable of stably expressing therapeutic molecules. Additionally, single cell cloning of these production lines is also necessary to confirm clonality of the production lines. Here we introduce the utilization of viability staining dye in combination with flow cytometer to isolate high titer clones from a pool of selected cells and single cell deposit them into the wells of culture plates. Our data suggests that a stringent selection procedure along with viability dye staining and flow cytometry-based sorting can be used to isolate high expressing clones with titers comparable to that of traditional CLD methods. This approach not only requires less labor and consumables, but it also shortens CLD timelines by at least 3 weeks. Furthermore, single cell deposition of selected cells by a flow sorter can be regarded as an additional clonality assurance factor that in combination with Day 0 imaging can ensure clonality of the production lines.

  16. Laser flow microphotometry for rapid analysis and sorting of mammalian cells. [X and gamma radiation

    Energy Technology Data Exchange (ETDEWEB)

    Mullaney, P.F.; Steinkamp, J.A.; Crissman, H.A.; Cram, L.S.; Crowell, J.M.; Salzman, G.C.; Martin, J.C.; Price, B.

    1976-01-01

    Quantitative precision measurements can be made of the optical properties of individual mammalian cells using flow microphotometry. Suspended cells pass through a special flow chamber where they are lined up for exposure to blue light from an argon-ion laser. As each cell crosses the laser beam, it produces one or more optical pulses of a duration equal to cell transit time across the beam. These pulses are detected, amplified, and analyzed using the techniques of gamma ray spectroscopy. Quantitative DNA distributions made it possible to distinguish tumor cells from normal cells as well as to assay for radiation effects on tumor cells subjected to x and gamma radiation. (HLW)

  17. Activation cross-section measurement of a sort of nuclide produced with a target including two isotopes

    Institute of Scientific and Technical Information of China (English)

    ZHOU Feng-Qun; TIAN Ming-Li; SONG Yue-Li; LAN Chang-Lin; KONG Xiang-Zhong

    2013-01-01

    Based on a formula used to calculate the activation cross-section sum of two reactions producing a sort of nuclide with a target including two isotopes,the related problems in some references have been analyzed and discussed.It is pointed out that the calculation methods of the cross-section sum of two reactions producing the same radioactive nuclide for two isotopes in some references are improper and usually it is impossible to obtain the correct cross-section sum of two reactions producing the same radioactive nuclide for two isotopes in the case of using natural samples.At the same time,the related concepts are clarified and the correct processing method and representation are given.The comparison with the experimental results show that the theoretical analysis results are right.

  18. New Approach to Selective Stem Cell Sorting: Separation of Undifferentiated Stem Cells from Differentiated Stem Cells by Using Iron Oxide Core Shell Nanoparticles

    Science.gov (United States)

    Kisa, Fikrullah

    An alternative approach to stem cell enrichment in another words sorting methods without changing the microenvironment of the cells to avoid the detrimental effects of present cell sorting methods by adopting iron-oxide gold (cFeAu) core-shell nanoparticles (NPs) is the focus of this thesis. Each chapter of this thesis focuses on different preliminary research in order to engender the adoption of cFeAu NPs for the selective killing of the mouse embryonic stem cells that are immunolabeled with the nanoparticles. The first part of the research focuses on the synthesis of superparamagnetic iron-oxide nanoparticles with the co-precipitation method and coating the nanoparticles with colloidal gold (cAu) to stabilize the characteristics of the nanoparticles. Detailed information regarding the chemistry of iron-oxide nanoparticles, common synthesis methods, and some of the factors that affect nanoparticle growth and synthesis have been investigated. The heating ability of the nanoparticles under an oscillating magnetic field (OMF) and the size distribution of the particles under a transmission electron microscope (TEM) are shown. The second part of the research focuses on selectively killing the RAW 264.7 macrophages which have internalized the synthesized nanoparticles in order to prove the biocompatibility and effectiveness of the nanoparticles. The particles' effect on the cells, the mechanism of killing, and the effectiveness of nanoparticles coated with colloidal gold and bovine serum albumin are investigated. The last part of the research focuses on effectively labeling the mESCs with a specific antibody conjugated to cFeAu nanoparticles that has an affinity to stage specific embryonic antigen 1 (SSEA-1). The influence of the OMF and the effects of immunolabeling on cell growth were investigated. The successful conjugation of the nanoparticles onto the cell surface is shown under scanning electron microscope. The damage inflicted by the nanoparticles on the cells

  19. [Physical arrangement of membrane lipids susceptible to being used in the process of cell sorting of proteins].

    Science.gov (United States)

    Wolf, C; Quinn, P; Koumanov, K; Chachaty, C; Tenchov, B

    1999-01-01

    Detection of immiscible lipid domains in biological membranes offers an alternative support to protein sorting. Liquid ordered domains ("rafts") comprising cholesterol and saturated sphingolipids incorporate saturated glycosyl-phosphatidylinositol (GPI)-anchored or acylated (palmitoyl- and myristoyl-) proteins or particular transmembrane protein sequences. These lipid domains can be isolated in the form of Detergent resistant membranes (DRM) from biological plasma membrane preparations. Caveolae appear to be a differentiated fraction of plasma membranes comprising such numerous cross-linked microdomains associated with caveolin in different cell types. While the biological relevance of such membrane domains is evidenced in vivo by co-patching of proteins sharing the identical affinity for sphingolipids and by the disruption of co-patching following cell cholesterol depletion, only a few physical studies confort the principle of membrane heterogeneity. Results are now presented where cholesterol addition in a tertiary lipid mixture forces outphase-separation, as a realistic model where the lipid segregation can promote protein sorting to the segregated Lo phase. A lipid mixture comprising phosphatidylserine, phosphatidylethanolamine and sphingomyelin of natural origin in the ratio (1/4/3: mole/mole) has been rendered neatly heterogeneous after the addition of cholesterol (27 mole%). Xray diffraction (Small angle Xray scattering) showed the splitting of two neatly resolved lamellar diffractions in the presence of cholesterol. Above 37 degrees C the heterogeneity was traceable by a broadened diffraction spot up to the complete get-to-liquid transition of sphingomyelin at temperatures > 40 degrees C where the spot became again symmetrical and narrow. The large temperature range where the immiscible lamellar phases are detected, the specific requirement for cholesterol association with sphingomyelin, the positive influence of calcium and the reversibility of domain

  20. Automated Chemotactic Sorting and Single-cell Cultivation of Microbes using Droplet Microfluidics

    National Research Council Canada - National Science Library

    Dong, Libing; Chen, Dong-Wei; Liu, Shuang-Jiang; Du, Wenbin

    2016-01-01

    ...; in the second part, chemotactic cells from the sample were separated, and mixed with culture media to form nanoliter droplets for encapsulation, cultivation, enumeration, and recovery of single cells...

  1. Ploidy of cell-sorted trophic and cystic forms of Pneumocystis carinii.

    Science.gov (United States)

    Martinez, Anna; Aliouat, El Moukhtar; Standaert-Vitse, Annie; Werkmeister, Elisabeth; Pottier, Muriel; Pinçon, Claire; Dei-Cas, Eduardo; Aliouat-Denis, Cécile-Marie

    2011-01-01

    Once regarded as an AIDS-defining illness, Pneumocystis pneumonia (PcP) is nowadays prevailing in immunocompromised HIV-negative individuals such as patients receiving immunosuppressive therapies or affected by primary immunodeficiency. Moreover, Pneumocystis clinical spectrum is broadening to non-severely-immunocompromised subjects who could be colonized by the fungus while remaining asymptomatic for PcP, thus being able to transmit the infection by airborne route to susceptible hosts. Although the taxonomical position of the Pneumocystis genus has been clarified, several aspects of its life cycle remain elusive such as its mode of proliferation within the alveolus or its ploidy level. As no long-term culture model exists to grow Pneumocystis organisms in vitro, an option was to use a model of immunosuppressed rat infected with Pneumocystis carinii and sort life cycle stage fractions using a high-through-put cytometer. Subsequently, ploidy levels of the P. carinii trophic and cystic form fractions were measured by flow cytometry. In the cystic form, eight contents of DNA were measured thus strengthening the fact that each mature cyst contains eight haploid spores. Following release, each spore evolves into a trophic form. The majority of the trophic form fraction was haploid in our study. Some less abundant trophic forms displayed two contents of DNA indicating that they could undergo (i) mating/fusion leading to a diploid status or (ii) asexual mitotic division or (iii) both. Even less abundant trophic forms with four contents of DNA were suggestive of mitotic divisions occurring following mating in diploid trophic forms. Of interest, was the presence of trophic forms with three contents of DNA, an unusual finding that could be related to asymmetrical mitotic divisions occurring in other fungal species to create genetic diversity at lower energetic expenses than mating. Overall, ploidy data of P. carinii life cycle stages shed new light on the complexity of its

  2. Ploidy of cell-sorted trophic and cystic forms of Pneumocystis carinii.

    Directory of Open Access Journals (Sweden)

    Anna Martinez

    Full Text Available Once regarded as an AIDS-defining illness, Pneumocystis pneumonia (PcP is nowadays prevailing in immunocompromised HIV-negative individuals such as patients receiving immunosuppressive therapies or affected by primary immunodeficiency. Moreover, Pneumocystis clinical spectrum is broadening to non-severely-immunocompromised subjects who could be colonized by the fungus while remaining asymptomatic for PcP, thus being able to transmit the infection by airborne route to susceptible hosts. Although the taxonomical position of the Pneumocystis genus has been clarified, several aspects of its life cycle remain elusive such as its mode of proliferation within the alveolus or its ploidy level. As no long-term culture model exists to grow Pneumocystis organisms in vitro, an option was to use a model of immunosuppressed rat infected with Pneumocystis carinii and sort life cycle stage fractions using a high-through-put cytometer. Subsequently, ploidy levels of the P. carinii trophic and cystic form fractions were measured by flow cytometry. In the cystic form, eight contents of DNA were measured thus strengthening the fact that each mature cyst contains eight haploid spores. Following release, each spore evolves into a trophic form. The majority of the trophic form fraction was haploid in our study. Some less abundant trophic forms displayed two contents of DNA indicating that they could undergo (i mating/fusion leading to a diploid status or (ii asexual mitotic division or (iii both. Even less abundant trophic forms with four contents of DNA were suggestive of mitotic divisions occurring following mating in diploid trophic forms. Of interest, was the presence of trophic forms with three contents of DNA, an unusual finding that could be related to asymmetrical mitotic divisions occurring in other fungal species to create genetic diversity at lower energetic expenses than mating. Overall, ploidy data of P. carinii life cycle stages shed new light on the

  3. Rab5 activity regulates GLUT4 sorting into insulin-responsive and non-insulin-responsive endosomal compartments: a potential mechanism for development of insulin resistance.

    Science.gov (United States)

    Tessneer, Kandice L; Jackson, Robert M; Griesel, Beth A; Olson, Ann Louise

    2014-09-01

    Glucose transporter isoform 4 (GLUT4) is the insulin-responsive glucose transporter mediating glucose uptake in adipose and skeletal muscle. Reduced GLUT4 translocation from intracellular storage compartments to the plasma membrane is a cause of peripheral insulin resistance. Using a chronic hyperinsulinemia (CHI)-induced cell model of insulin resistance and Rab5 mutant overexpression, we determined these manipulations altered endosomal sorting of GLUT4, thus contributing to the development of insulin resistance. We found that CHI induced insulin resistance in 3T3-L1 adipocytes by retaining GLUT4 in a Rab5-activity-dependent compartment that is unable to equilibrate with the cell surface in response to insulin. Furthermore, CHI-mediated retention of GLUT4 in this non-insulin-responsive compartment impaired filling of the transferrin receptor (TfR)-positive and TfR-negative insulin-responsive storage compartments. Our data suggest that hyperinsulinemia may inhibit GLUT4 by chronically maintaining GLUT4 in the Rab5 activity-dependent endosomal pathway and impairing formation of the TfR-negative and TfR-positive insulin-responsive GLUT4 pools. This model suggests that an early event in the development of insulin-resistant glucose transport in adipose tissue is to alter the intracellular localization of GLUT4 to a compartment that does not efficiently equilibrate with the cell surface when insulin levels are elevated for prolonged periods of time.

  4. A new principle of cell sorting by using selective electroporation in a modified flow cytometer

    NARCIS (Netherlands)

    Bakker Schut, Tom C.; Grooth, de Bart G.; Greve, Jan

    1990-01-01

    When a strong electric field pulse of a few microseconds is applied to biological cells, small pores are formed in the cell membranes; this process is called electroporation. At high field strengths and/or long pulse durations the membranes will be damaged permanently. This eventually leads to cell

  5. Isolation, cultivation and identification of brain glioma stem cells by magnetic bead sorting

    Institute of Scientific and Technical Information of China (English)

    Xiuping Zhou; Chao Zheng; Qiong Shi; Xiang Li; Zhigang Shen; Rutong Yu

    2012-01-01

    This study describes a detailed process for obtaining brain glioma stem cells from freshly dissected human brain glioma samples using an immunomagnetic bead technique combined with serum-free media pressure screening. Furthermore, the proliferation, differentiation and self-renewal biological features of brain glioma stem cells were identified. Results showed that a small number of CD133 positive tumor cells isolated from brain glioma samples survived as a cell suspension in serum-free media and proliferated. Subcultured CD133 positive cells maintained a potent self-renewal and proliferative ability, and expressed the stem cell-specific markers CD133 and nestin. After incubation with fetal bovine serum, the number of glial fibrillary acidic protein and microtubule associated protein 2 positive cells increased significantly, indicating that the cultured brain glioma stem cells can differentiate into astrocytes and neurons. Western blot analysis showed that tumor suppressor phosphatase and tensin homolog was highly expressed in tumor spheres compared with the differentiated tumor cells. These experimental findings indicate that the immunomagnetic beads technique is a useful method to obtain brain glioma stem cells from human brain tumors.

  6. Efficient Parallel Sorting for Migrating Birds Optimization When Solving Machine-Part Cell Formation Problems

    OpenAIRE

    Ricardo Soto; Broderick Crawford; Boris Almonacid; Fernando Paredes

    2016-01-01

    The Machine-Part Cell Formation Problem (MPCFP) is a NP-Hard optimization problem that consists in grouping machines and parts in a set of cells, so that each cell can operate independently and the intercell movements are minimized. This problem has largely been tackled in the literature by using different techniques ranging from classic methods such as linear programming to more modern nature-inspired metaheuristics. In this paper, we present an efficient parallel version of the Migrating Bi...

  7. T cell receptor zeta allows stable expression of receptors containing the CD3gamma leucine-based receptor-sorting motif

    DEFF Research Database (Denmark)

    Dietrich, J; Geisler, C

    1998-01-01

    The leucine-based motif in the T cell receptor (TCR) subunit CD3gamma constitutes a strong internalization signal. In fully assembled TCR this motif is inactive unless phosphorylated. In contrast, the motif is constitutively active in CD4/CD3gamma and Tac/CD3gamma chimeras independently of phosph......The leucine-based motif in the T cell receptor (TCR) subunit CD3gamma constitutes a strong internalization signal. In fully assembled TCR this motif is inactive unless phosphorylated. In contrast, the motif is constitutively active in CD4/CD3gamma and Tac/CD3gamma chimeras independently...... of phosphorylation and leads to rapid internalization and sorting of these chimeras to lysosomal degradation. Because the TCRzeta chain rescues incomplete TCR complexes from lysosomal degradation and allows stable surface expression of fully assembled TCR, we addressed the question whether TCRzeta has the potential...... to mask the CD3gamma leucine-based motif. By studying CD4/CD3gamma and CD16/CD3gamma chimeras, we found that CD16/CD3gamma chimeras associated with TCRzeta. The CD16/CD3gamma-TCRzeta complexes were stably expressed at the cell surface and had a low spontaneous internalization rate, indicating...

  8. Induction and repair of DNA damage measured by the comet assay in human T lymphocytes separated by immunomagnetic cell sorting.

    Science.gov (United States)

    Bausinger, Julia; Speit, Günter

    2014-11-01

    The comet assay is widely used in human biomonitoring to measure DNA damage in whole blood or isolated peripheral blood mononuclear cells (PBMC) as a marker of exposure to genotoxic agents. Cytogenetic assays with phytohemagglutinin (PHA)-stimulated cultured T lymphocytes are also frequently performed in human biomonitoring. Cytogenetic effects (micronuclei, chromosome aberrations, sister chromatid exchanges) may be induced in vivo but also occur ex vivo during the cultivation of lymphocytes as a consequence of DNA damage present in lymphocytes at the time of sampling. To better understand whether DNA damage measured by the comet assay in PBMC is representative for DNA damage in T cells, we comparatively investigated DNA damage and its repair in PBMC and T cells obtained by immunomagnetic cell sorting. PBMC cultures and T cell cultures were exposed to mutagens with different modes of genotoxic action and DNA damage was measured by the comet assay after the end of a 2h exposure and after 18h post-incubation. The mutagens tested were methyl methanesulfonate (MMS), (±)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), 4-nitroquinoline-1-oxide (4NQO), styrene oxide and potassium bromate. MMS and potassium bromate were also tested by the modified comet assay with formamido pyrimidine glycosylase (FPG) protein. The results indicate that the mutagens tested induce DNA damage in PBMC and T cells in the same range of concentrations and removal of induced DNA lesions occurs to a comparable extent. Based on these results, we conclude that the comet assay with PBMC is suited to predict DNA damage and its removal in T cells.

  9. An improved cell separation technique for marine subsurface sediments: applications for high-throughput analysis using flow cytometry and cell sorting.

    Science.gov (United States)

    Morono, Yuki; Terada, Takeshi; Kallmeyer, Jens; Inagaki, Fumio

    2013-10-01

    Development of an improved technique for separating microbial cells from marine sediments and standardization of a high-throughput and discriminative cell enumeration method were conducted. We separated microbial cells from various types of marine sediment and then recovered the cells using multilayer density gradients of sodium polytungstate and/or Nycodenz, resulting in a notably higher percent recovery of cells than previous methods. The efficiency of cell extraction generally depends on the sediment depth; using the new technique we developed, more than 80% of the total cells were recovered from shallow sediment samples (down to 100 meters in depth), whereas ~50% of cells were recovered from deep samples (100-365 m in depth). The separated cells could be rapidly enumerated using flow cytometry (FCM). The data were in good agreement with those obtained from manual microscopic direct counts over the range 10(4)-10(8) cells cm(-3). We also demonstrated that sedimentary microbial cells can be efficiently collected using a cell sorter. The combined use of our new cell separation and FCM/cell sorting techniques facilitates high-throughput and precise enumeration of microbial cells in sediments and is amenable to various types of single-cell analyses, thereby enhancing our understanding of microbial life in the largely uncharacterized deep subseafloor biosphere.

  10. Word Sorts for General Music Classes

    Science.gov (United States)

    Cardany, Audrey Berger

    2015-01-01

    Word sorts are standard practice for aiding children in acquiring skills in English language arts. When included in the general music classroom, word sorts may aid students in acquiring a working knowledge of music vocabulary. The author shares a word sort activity drawn from vocabulary in John Lithgow's children's book "Never Play…

  11. Hydrodynamic lift of vesicles and red blood cells in flow--from Fåhræus & Lindqvist to microfluidic cell sorting.

    Science.gov (United States)

    Geislinger, Thomas M; Franke, Thomas

    2014-06-01

    Hydrodynamic lift forces acting on cells and particles in fluid flow receive ongoing attention from medicine, mathematics, physics and engineering. The early findings of Fåhræus & Lindqvist on the viscosity change of blood with the diameter of capillaries motivated extensive studies both experimentally and theoretically to illuminate the underlying physics. We review this historical development that led to the discovery of the inertial and non-inertial lift forces and elucidate the origins of these forces that are still not entirely clear. Exploiting microfluidic techniques induced a tremendous amount of new insights especially into the more complex interactions between the flow field and deformable objects like vesicles or red blood cells. We trace the way from the investigation of single cell dynamics to the recent developments of microfluidic techniques for particle and cell sorting using hydrodynamic forces. Such continuous and label-free on-chip cell sorting devices promise to revolutionize medical analyses for personalized point-of-care diagnosis. We present the state-of-the-art of different hydrodynamic lift-based techniques and discuss their advantages and limitations.

  12. Parallel optical sorting of biological cells using the generalized phase contrast method

    DEFF Research Database (Denmark)

    Rindorf, Lars; Bu, Minqiang; Glückstad, Jesper

    2014-01-01

    of biological cells in microfluidic systems exclusively using light. We demonstrate an optical cell sorter that uses simultaneous manipulation by multiple laser beams using the Generalized Phase Contrast method (GPC). The basic principle in an optical sorter is that the radiation force of the optical beam can...... push the biological cell from one microfluidic sheath flow to another. By incorporating a spatial light modulator the manipulation can be made parallel with multiple laser beams. We claim advantages over the serial optical sorters with only a single laser beam that has been demonstrated by others.......Optical forces are used to fixate biological cells with optical tweezers where numerous biological parameters and phenomena can be studied. Optical beams carry a small momentum which generates a weak optical force, but on a cellular level this force is strong enough to allow for manipulation...

  13. Laser applications and anticipated laser requirements in rapid analysis and sorting of mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Cram, L.S.; Crissman, H.A.; Martin, J.C.; Price, B.J.; Salzman, G.C.; Steinkamp, J.A.

    1976-01-01

    The rapidly expanding field of fast flow-cell analysis is making contributions to almost all areas of biology and medicine. Basic biology is profitting greatly from quantitative information and the availability of pure cell populations heretofore unavailable. Clinical applications utilizing fast flow analysis for diagnostic medicine are appearing and, as a result, commercial firms have entered the field to market instruments designed for different applications. Future developments will certainly be important in bacterial mutant selection, in isolation of new subpopulations of cells involved in the immune system, and in cancer-cell screening where the high throughput capabilities of the instrumentation can be utilized best. Extremely small beam spots on the order of 0.5 to 1 ..mu..m and the ability to sweep such spots very rapidly across the surface of a cell would be very advantageous for obtaining morphologic information, instead of the standard bulk (or whole-cell) information. New biological questions are arising that will necessitate using pulsed lasers to look at fluorescence decay times.

  14. Specialized sorting of GLUT4 and its recruitment to the cell surface are independently regulated by distinct Rabs.

    Science.gov (United States)

    Sadacca, L Amanda; Bruno, Joanne; Wen, Jennifer; Xiong, Wenyong; McGraw, Timothy E

    2013-08-01

    Adipocyte glucose uptake in response to insulin is essential for physiological glucose homeostasis: stimulation of adipocytes with insulin results in insertion of the glucose transporter GLUT4 into the plasma membrane and subsequent glucose uptake. Here we establish that RAB10 and RAB14 are key regulators of GLUT4 trafficking that function at independent, sequential steps of GLUT4 translocation. RAB14 functions upstream of RAB10 in the sorting of GLUT4 to the specialized transport vesicles that ferry GLUT4 to the plasma membrane. RAB10 and its GTPase-activating protein (GAP) AS160 comprise the principal signaling module downstream of insulin receptor activation that regulates the accumulation of GLUT4 transport vesicles at the plasma membrane. Although both RAB10 and RAB14 are regulated by the GAP activity of AS160 in vitro, only RAB10 is under the control of AS160 in vivo. Insulin regulation of the pool of RAB10 required for GLUT4 translocation occurs through regulation of AS160, since activation of RAB10 by DENND4C, its GTP exchange factor, does not require insulin stimulation.

  15. Isolation of carotenoid hyperproducing mutants of Xanthophyllomyces dendrorhous (Phaffia rhodozyma) by flow cytometry and cell sorting.

    Science.gov (United States)

    Brehm-Stecher, Byron F; Johnson, Eric A

    2012-01-01

    Approaches for improving astaxanthin yields in Xanthophyllomyces dendrorhous include optimization of fermentation conditions and generation of hyperproducing mutants through random mutagenesis using chemical or physical means. A key limitation of classical mutagenesis is the labor-intensive nature of the screening processes required to find relatively rare mutants having increased carotenoid content, as these are present against a high background of low-interest cells. Here, flow cytometry is described as a high-throughput, single-cell method for primary enrichment of mutagenized cells expressing high levels of astaxanthin. This approach improves the speed and productivity of classical strain selection, enhancing the chances for isolating the carotenoid hyperproducing mutants (CHMs) needed to enable high-titer, economical production of natural astaxanthin.

  16. ProSAAS-derived peptides are differentially processed and sorted in mouse brain and AtT-20 cells.

    Directory of Open Access Journals (Sweden)

    Jonathan H Wardman

    Full Text Available ProSAAS is the precursor for some of the most abundant peptides found in mouse brain and other tissues, including peptides named SAAS, PEN, and LEN. Both SAAS and LEN are found in big and little forms due to differential processing. Initial processing of proSAAS is mediated by furin (and/or furin-like enzymes and carboxypeptidase D, while the smaller forms are generated by secretory granule prohormone convertases and carboxypeptidase E. In mouse hypothalamus, PEN and big LEN colocalize with neuropeptide Y. In the present study, little LEN and SAAS were detected in mouse hypothalamus but not in cell bodies of neuropeptide Y-expressing neurons. PEN and big LEN show substantial colocalization in hypothalamus, but big LEN and little LEN do not. An antiserum to SAAS that detects both big and little forms of this peptide did not show substantial colocalization with PEN or big LEN. To further study this, the AtT-20 cells mouse pituitary corticotrophic cell line was transfected with rat proSAAS and the distribution of peptides examined. As found in mouse hypothalamus, only some of the proSAAS-derived peptides colocalized with each other in AtT-20 cells. The two sites within proSAAS that are known to be efficiently cleaved by furin were altered by site-directed mutagenesis to convert the P4 Arg into Lys; this change converts the sequences from furin consensus sites into prohormone convertase consensus sites. Upon expression of the mutated form of proSAAS in AtT-20 cells, there was significantly more colocalization of proSAAS-derived peptides PEN and SAAS. Taken together, these results indicate that proSAAS is initially cleaved in the Golgi or trans-Golgi network by furin and/or furin-like enzymes and the resulting fragments are sorted into distinct vesicles and further processed by additional enzymes into the mature peptides.

  17. Characterization of heterotrophic prokaryote subgroups in the Sfax coastal solar salterns by combining flow cytometry cell sorting and phylogenetic analysis.

    Science.gov (United States)

    Trigui, Hana; Masmoudi, Salma; Brochier-Armanet, Céline; Barani, Aude; Grégori, Gérald; Denis, Michel; Dukan, Sam; Maalej, Sami

    2011-05-01

    Here, we combined flow cytometry (FCM) and phylogenetic analyses after cell sorting to characterize the dominant groups of the prokaryotic assemblages inhabiting two ponds of increasing salinity: a crystallizer pond (TS) with a salinity of 390 g/L, and the non-crystallizer pond (M1) with a salinity of 200 g/L retrieved from the solar saltern of Sfax in Tunisia. As expected, FCM analysis enabled the resolution of high nucleic acid content (HNA) and low nucleic acid content (LNA) prokaryotes. Next, we performed a taxonomic analysis of the bacterial and archaeal communities comprising the two most populated clusters by phylogenetic analyses of 16S rRNA gene clone library. We show for the first time that the presence of HNA and LNA content cells could also be extended to the archaeal populations. Archaea were detected in all M1 and TS samples, whereas representatives of Bacteria were detected only in LNA for M1 and HNA for TS. Although most of the archaeal sequences remained undetermined, other clones were most frequently affiliated to Haloquadratum and Halorubrum. In contrast, most bacterial clones belonged to the Alphaproteobacteria class (Phyllobacterium genus) in M1 samples and to the Bacteroidetes phylum (Sphingobacteria and Salinibacter genus) in TS samples.

  18. How are ‘atypical’ sulfite dehydrogenases linked to cell metabolism? – Interactions between the SorT sulfite deydrogenase and small redox proteins

    Directory of Open Access Journals (Sweden)

    Louie eLow

    2011-03-01

    Full Text Available Sulfite dehydrogenases are enzymes that catalyze the oxidation of the toxic and mutagenic compound sulfite to sulfate, thereby protecting cells from adverse effects associated with sulfite exposure. While some bacterial sulfite dehydrogenases that have been characterized to date are able to use cytochrome c as an electron acceptor, the majority of these enzymes prefer ferricyanide as an electron acceptor and have therefore been termed ‘atypical’ sulfite dehydrogenases. Identifying the natural electron acceptor of these enzymes, however, is crucial for understanding how the ‘atypical’ sulfite dehydrogenases are integrated into cell metabolism.The SorT sulfite dehydrogenase from Sinorhizobium meliloti is a representative of this enzyme type and we have investigated the interactions of SorT with two small redox proteins, a cytochrome c and a Cu containing pseudoazurin, that are encoded in the same operon and are co-transcribed with the sorT gene. Both potential acceptor proteins have been purified and characterized in terms of their biochemical and electrochemical properties, and interactions and enzymatic studies with both the purified SorT sulfite dehydrogenase and components of the respiratory chain have been carried out.We were able to show for the first time that an ‘atypical’ sulfite dehydrogenase can couple efficiently to a cytochrome c isolated from the same organism despite being unable to efficiently reduce horse heart cytochrome c, however, at present the role of the pseudoazurin in SorT electron transfer is unclear, but it is possible that it acts as an intermediate electron shuttle between. The SorT system appears to couple directly to the respiratory chain, most likely to a cytochrome oxidase.

  19. Towards microfluidic sperm refinement : impedance-based analysis and sorting of sperm cells

    NARCIS (Netherlands)

    Wagenaar, de B.; Dekker, S.; Boer, de H.L.; Bomer, J.G.; Olthuis, W.; Berg, van den A.; Segerink, L.I.

    2016-01-01

    The use of high quality semen for artificial insemination in the livestock industry is essential for successful outcome. Insemination using semen with a high number of sperm cells containing morphological defects has a negative impact on fertilization outcome. Therefore, semen with a high number of

  20. Detection of internal structure by scattered light intensity: Application to kidney cell sorting

    Science.gov (United States)

    Goolsby, C. L.; Kunze, M. E.

    1985-01-01

    Scattered light measurements in flow cytometry were sucessfully used to distinguish cells on the basis of differing morphology and internal structure. Differences in scattered light patterns due to changes in internal structure would be expected to occur at large scattering angles. Practically, the results of these calculations suggest that in experimental situations an array of detectors would be useful. Although in general the detection of the scattered light intensity at several intervals within the 10 to 60 region would be sufficient, there are many examples where increased sensitivity could be acheived at other angles. The ability to measure at many different angular intervals would allow the experimenter to empirically select the optimum intervals for the varying conditions of cell size, N/C ratio, granule size and internal structure from sample to sample. The feasibility of making scattered light measurements at many different intervals in flow cytometry was demonstrated. The implementation of simplified versions of these techniques in conjunction with independant measurements of cell size could potentially improve the usefulness of flow cytometry in the study of the internal structure of cells.

  1. Towards microfluidic sperm refinement : impedance-based analysis and sorting of sperm cells

    NARCIS (Netherlands)

    de Wagenaar, B.; Dekker, Stefan; de Boer, Hans L.; Bomer, Johan G.; Olthuis, Wouter; van den Berg, Albert; Segerink, Loes Irene

    2016-01-01

    The use of high quality semen for artificial insemination in the livestock industry is essential for successful outcome. Insemination using semen with a high number of sperm cells containing morphological defects has a negative impact on fertilization outcome. Therefore, semen with a high number of

  2. Sorting Nexin 27 Regulates Aβ Production through Modulating γ-Secretase Activity

    Directory of Open Access Journals (Sweden)

    Xin Wang

    2014-11-01

    Full Text Available Patients with Down syndrome (DS invariably develop Alzheimer’s disease (AD pathology in their 40s. We have recently found that overexpression of a chromosome 21-encoded microRNA-155 results in decreased levels of the membrane trafficking component, SNX27, diminishing glutamate receptor recycling and thereby impairing synaptic functions in DS. Here, we report a function of SNX27 in regulating β-amyloid (Aβ generation by modulating γ-secretase activity. Downregulation of SNX27 using RNAi increased Aβ production, whereas overexpression of full-length SNX27, but not SNX27ΔPDZ, reversed the RNAi-mediated Aβ elevation. Moreover, genetic deletion of Snx27 promoted Aβ production and neuronal loss, whereas overexpression of SNX27 using an adeno-associated viral (AAV vector reduced hippocampal Aβ levels in a transgenic AD mouse model. SNX27 associates with the γ-secretase complex subunit presenilin 1; this interaction dissociates the γ-secretase complex, thus decreasing its proteolytic activity. Our study establishes a molecular mechanism for Aβ-dependent pathogenesis in both DS and AD.

  3. Design of a Microfluidic Chip for Magnetic-Activated Sorting of One-Bead-One-Compound Libraries.

    Science.gov (United States)

    Cho, Choi-Fong; Lee, Kyungheon; Speranza, Maria-Carmela; Bononi, Fernanda C; Viapiano, Mariano S; Luyt, Leonard G; Weissleder, Ralph; Chiocca, E Antonio; Lee, Hakho; Lawler, Sean E

    2016-06-13

    Molecular targeting using ligands specific to disease markers has shown great promise for early detection and directed therapy. Bead-based combinatorial libraries have served as powerful tools for the discovery of novel targeting agents. Screening platforms employing magnetic capture have been used to achieve rapid and efficient identification of high-affinity ligands from one-bead-one-compound (OBOC) libraries. Traditional manual methodologies to isolate magnetized "hit" beads are tedious and lack accuracy, and existing instruments to expedite bead sorting tend to be costly and complex. Here, we describe the design and construction of a simple and inexpensive microfluidic magnetic sorting device using standard photolithography and soft lithography approaches to facilitate high-throughput isolation of magnetized positive hit beads from combinatorial libraries. We have demonstrated that the device is able to sort magnetized beads with superior accuracy compared to conventional manual sorting approaches. This chip offers a very convenient yet inexpensive alternative for screening OBOC libraries.

  4. Progress in Analytical Techniques of Microfluidic Chip on Cell Sorting%微流控芯片在细胞分选中的分析技术进展

    Institute of Scientific and Technical Information of China (English)

    刘琳; 厉坤鹏; 胡志敏; 董军磊; 欧元; 李彩霞; 赵兴春; 叶健

    2013-01-01

    微流控芯片分析技术以其快速、高效、高能量、低消耗、集成化和微型化等特点在多个研究领域发展非常迅速.该文根据分选原理不同,将微流控芯片上的细胞分选方法分为主动式细胞分选与被动式细胞分选,从这两方面总结了目前微芯片分选细胞的进展,并对该技术在细胞分选中的应用前景作了进一步的展望.%Microfluidic chip techniques develop very fast owing to it's high rate,high efficiency,high power,low reagent cost,integrating and miniaturization in many research fields.Based on the operating principles,the various cell sorting methods on microfluidic chip are broadly categorized as active or passive techniques in this article.Recent research progress of microfluidic chip techniques on cell sorting are summarized mainly in the two aspects.In addition,the application prospect of microfluidic chip on cell sorting is also briefly discussed.

  5. Sortilin regulates sorting and secretion of Sonic hedgehog.

    Science.gov (United States)

    Campbell, Charles; Beug, Shawn; Nickerson, Philip E B; Peng, Jimmy; Mazerolle, Chantal; Bassett, Erin A; Ringuette, Randy; Jama, Fadumo A; Morales, Carlos; Christ, Annabel; Wallace, Valerie A

    2016-10-15

    Sonic Hedgehog (Shh) is a secreted morphogen that is an essential regulator of patterning and growth. The Shh full-length protein undergoes autocleavage in the endoplasmic reticulum to generate the biologically active N-terminal fragment (ShhN), which is destined for secretion. We identified sortilin (Sort1), a member of the VPS10P-domain receptor family, as a new Shh trafficking receptor. We demonstrate that Sort-Shh interact by performing coimmunoprecipitation and proximity ligation assays in transfected cells and that they colocalize at the Golgi. Sort1 overexpression causes re-distribution of ShhN and, to a lesser extent, of full-length Shh to the Golgi and reduces Shh secretion. We show loss of Sort1 can partially rescue Hedgehog-associated patterning defects in a mouse model that is deficient in Shh processing, and we show that Sort1 levels negatively regulate anterograde Shh transport in axons in vitro and Hedgehog-dependent axon-glial interactions in vivo Taken together, we conclude that Shh and Sort1 can interact at the level of the Golgi and that Sort1 directs Shh away from the pathways that promote its secretion.

  6. k -Bitonic sort

    Institute of Scientific and Technical Information of China (English)

    高庆狮; 胡玥; 刘志勇

    1999-01-01

    A k-bitonic sort which generalizes the bitonic sort is proposed. The theorem of the bitonic sort, which merges two monotonic sequences into one order sequence, is extended into the theorem of k-bitonic sort. The k-bitonic sort merges K (=2k or 2k-1) monotonic sequences into one order sequence in steps, where k=[K/2] is an integer and k≥1. The k-bitonic sort is the Batcher’s bitonic sort when k=1.

  7. Enrichment of fetal cells from maternal blood by high gradient magnetic cell sorting (double MACS) for PCR-based genetic analysis.

    Science.gov (United States)

    Büsch, J; Huber, P; Pflüger, E; Miltenyi, S; Holtz, J; Radbruch, A

    1994-12-01

    For simple and effective isolation of fetal cells from peripheral maternal blood, we combined depletion of maternal cells and enrichment of fetal cells by high-gradient magnetic cell separation (MACS). First CD45+ and CD14+ cells were depleted from maternal peripheral blood mononuclear cells by MACS. From the depleted fraction, CD71+ erythroid cells were enriched up to 80 per cent by MACS. This double-MACS' procedure yielded an average depletion rate of 780-fold and an average enrichment rate of 500-fold, with approximate recovery rates of 40-55 per cent. For paternity testing, cells from unseparated blood and the various fractions were analysed for polymorphism of the HLA-DQ-A1 locus and D1S80 locus by the polymerase chain reaction (PCR). In CD45-/CD71+ sorted cells from maternal blood, but not in unfractionated cells from maternal blood or CD45-/CD14- cells, paternal alleles could be detected. In the CD45-/CD71+ fraction, the relative frequency of paternal alleles compared with maternal alleles ranged from 1 in 20 to 1 in 200 (determined by titration and depending on the quality of separation and biological variation). In 7 out of 11 cases, between weeks 12 and 25 of gestation, we could identify paternal alleles by PCR, either HLA-DQ-A1 or D1S80. This double-MACS procedure is simple, fast, efficient, and reliable for non-invasive prenatal diagnosis.

  8. Driving gradual endogenous c-myc overexpression by flow-sorting: intracellular signaling and tumor cell phenotype correlate with oncogene expression

    DEFF Research Database (Denmark)

    Knudsen, Kasper Jermiin; Holm, G.M.N.; Krabbe, J.S.

    2009-01-01

    cells than in the nonsorted cell population. To our knowledge, this is the first in vitro system allowing functional coupling between mitogenic signaling by a well-defined growth factor and gradual overexpression of the normal, endogenous c-myc gene. Thus, our flow-sorting approach provides...... an alternative modeling of the receptor-mediated carcinogenic process, compared to the currently used approaches of recombinant constitutive or conditional overexpression of oncogenic transmembrane receptor tyrosine kinases or oncogenic transcription factors....

  9. Optical force on diseased blood cells: Towards the optical sorting of biological matter

    KAUST Repository

    Gongora, J. S. Totero

    2015-05-01

    By employing a series of massively parallel ab-initio simulations, we study how optical forces act on biological matter subject to morphological disease. As a representative case study, we here consider the case of Plasmodium falciparum on red blood cells (RBC) illuminated by a monochromatic plane wave. Realistic parameters for the geometry and the refractive index are then taken from published experiments. In our theoretical campaign, we study the dependence of the optical force on the disease stage for different incident wavelengths. We show that optical forces change significantly with the disease, with amplitude variation in the hundreds of pN range. Our results open up new avenues for the design of new optical systems for the treatment of human disease. © 2015 Elsevier Ltd.

  10. Optical force on diseased blood cells: towards the optical sorting of biological matter

    CERN Document Server

    Gongora, Juan Sebastian Totero

    2016-01-01

    By employing a series of massively parallel ab-initio simulations, we study how optical forces act on biological matter subject to morphological disease. As a representative case study, we here consider the case of Plasmodium Falciparum on red blood cells (RBC) illuminated by a monochromatic plane wave. Realistic parameters for the geometry and the refractive index are then taken from published experiments. In our theoretical campaign, we study the dependence of the optical force on the disease stage for different incident wavelengths. We show that optical forces change significantly with the disease, with amplitude variation in the hundreds of pN range. Our results open up new avenues for the design of new optical systems for the treatment of human disease.

  11. Apposition of iroquois expressing and non-expressing cells leads to cell sorting and fold formation in the Drosophila imaginal wing disc

    Directory of Open Access Journals (Sweden)

    González-Pérez Esther

    2007-09-01

    Full Text Available Abstract Background The organization of the different tissues of an animal requires mechanisms that regulate cell-cell adhesion to promote and maintain the physical separation of adjacent cell populations. In the Drosophila imaginal wing disc the iroquois homeobox genes are expressed in the notum anlage and contribute to the specification of notum identity. These genes are not expressed in the adjacent wing hinge territory. These territories are separated by an approximately straight boundary that in the mature disc is associated with an epithelial fold. The mechanism by which these two cell populations are kept separate is unclear. Results Here we show that the Iro-C genes participate in keeping the notum and wing cell populations separate. Indeed, within the notum anlage, cells not expressing Iro-C tend to join together and sort out from their Iro-C expressing neighbours. We also show that apposition of Iro-C expressing and non-expressing cells induces invagination and apico-basal shortening of the Iro-C- cells. This effect probably underlies formation of the fold that separates the notum and wing hinge territories. In addition, cells overexpressing a member of the Iro-C contact one another and become organized in a network of thin strings that surrounds and isolates large groups of non-overexpressing cells. The strings appear to exert a pulling force along their longitudinal axis. Conclusion Apposition of cells expressing and non-expressing the Iro-C, as it occurs in the notum-wing hinge border of the Drosophila wing disc, influences cell behaviour. It leads to cell sorting, and cellular invagination and apical-basal shortening. These effects probably account for keeping the prospective notum and wing hinge cell populations separate and underlie epithelial fold formation. Cells that overexpress a member of the Iro-C and that confront non-expressing cells establish contacts between themselves and become organized in a network of thin strings

  12. Preoperative sorting of circulating T lymphocytes in patients with esophageal squamous cell carcinoma: Its prognostic significance

    Institute of Scientific and Technical Information of China (English)

    Tadahiro Nozoe; Yoshihiko Maehara; Keizo Sugimachi

    2005-01-01

    AIM: To elucidate the immunologic parameters for the outcome of patients with malignant tumors, especially esophageal squamous cell carcinoma (ESCC) associated with high malignant potential.METHODS: Clinicopathologic features were compared between patients with lower and higher CD4 and CD8values as well as CD4/CD8 ratio in peripheral blood.RESULTS: The survival rate of patients with higher CD4 value was significantly better than that in patients with lower CD4 value (P = 0.039). The survival rate of patients with higher CD8 value was significantly worse than that of patients with lower CD8 value (P = 0.026).Similarly, the survival rate of patients with higher CD4/CD8 ratio was significantly better than that of patients with lower CD4/CD8 ratio (P = 0.042). Additionally,multivariate analysis demonstrated that lower CD8and lower CD4/CD8 ratio were factors independently associated with worse prognosis of patients.CONCLUSION: All the immunologic parameters can predict the outcome of patients with ESCC.

  13. The C-terminal domain of zDHHC2 contains distinct sorting signals that regulate intracellular localisation in neurons and neuroendocrine cells.

    Science.gov (United States)

    Salaun, Christine; Ritchie, Louise; Greaves, Jennifer; Bushell, Trevor J; Chamberlain, Luke H

    2017-07-30

    The S-acyltransferase zDHHC2 mediates dynamic S-acylation of PSD95 and AKAP79/150, which impacts synaptic targeting of AMPA receptors. zDHHC2 is responsive to synaptic activity and catalyses the increased S-acylation of PSD95 that occurs following action potential blockade or application of ionotropic glutamate receptor antagonists. These treatments have been proposed to increase plasma membrane delivery of zDHHC2 via an endosomal cycling pathway, enhancing substrate accessibility. To generate an improved understanding of zDHHC2 trafficking and how this might be regulated by neuronal activity, we searched for intramolecular signals that regulate enzyme localisation. Two signals were mapped to the C-terminal tail of zDHHC2: a non-canonical dileucine motif [SxxxLL] and a downstream NP motif. Mutation of these signals enhanced plasma membrane accumulation of zDHHC2 in both neuroendocrine PC12 cells and rat hippocampal neurons, consistent with reduced endocytic retrieval. Furthermore, mutation of these signals also increased accumulation of the enzyme in neurites. Interestingly, several threonine and serine residues are adjacent to these sorting motifs and analysis of phospho-mimetic mutants highlighted a potential role for phosphorylation in regulating the efficacy of these signals. This study offers new molecular insight into the signals that determine zDHHC2 localisation and highlights a potential mechanism to regulate these trafficking signals. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Immunomagenetic indirect positive sorting of neural stem cells from fetal rat brain%免疫磁珠法分选胚胎大鼠脑神经干细胞的初步研究

    Institute of Scientific and Technical Information of China (English)

    高国一; 李莉

    2000-01-01

    目的:探索应用免疫磁珠间接阳性法分选神经上皮干细胞蛋白(nestin)阳性的神经干细胞群的实验条件.为研究神经干细胞的特性与神经干细胞的培养和移植研究创造有利条件。方法:制取胎鼠大脑组织细胞悬液,免疫磁珠法分选胎鼠脑神经干细胞,以流式细胞术检测阳性细胞纯度,以锥虫蓝染色法检测细胞活性。结果:该法分选的nestin阳性细胞纯度为93.0%~99.7%,其中活性细胞为92%~97%。结论:免疫磁珠法分离胎鼠脑神经干细胞群落简便、有效,可以为神经干细胞细胞培养和移植提供细胞来源,也为研究高纯度神经干细胞的特性提供了实验基础。%Objective:To study the sorting high-purity neural stem cells fromfetal rat brain cells.Methods:Fetal rat brain cell suspensions were incubated with monoclone antibody of nestin,and the labelled cells were separated from the suspension in the magnetic field by immunobeads coated with the second antibody.Purity of the sorted cells was determined with flow cytometry.Results:Purity of the sorted neural stem cells were determined as 93.0%-99.7%,active cells acount for 92 %-97 %.Conclusion:The magnetic cell sorting system can effectively separate neural stem cell from brain cell suspension.

  15. Expression of stem cell markers in side population cells sorted from SMMC-7721 cell line%肝癌SMMC-7721细胞中侧群细胞干细胞标记的表达

    Institute of Scientific and Technical Information of China (English)

    黄涛; 宫东伟; 高全立; 张旭华; 吕晓东; 周进学

    2011-01-01

    Objective To study the expression of stem cell markers in side population cells sorted from SMMC-7721 cell line. Methods Fluorescence-activated cell sorting (FACS) was used to sort side population (SP) cells and non-SP (NSP) cells from SMMC-7721 cell line. Real-time polymerase chain reaction (PCR) and flow cytometry (FCM) were used to evaluate the expression of several stem cell markers such as ABCG2, CD133, Oct4, Sox2 and NANOG in SP cells and NSP cells. Results FACS analysis indicated that (9.2 ±0. 2)% of the SMMC-7721 cells were SP cells. Real-time PCR analysis suggested that ABCG2, CD133, Oct4, Sox2 and NANOG were expressed in the SP cells at higher levels than the NSP cells by about 7. 132, 4. 985, 8. 642, 5.095 and 5. 164 folds, respectively ( P <0. 01 ). FCM analysis revealed that the expression of ABCG2, CD133, Oct4, Sox2 and NANOG proteins in SP cells was (92. 65 ±3.92)%, (12.75 ±1.62)%, (17.35 ±2.31)%, (9.57 ± 1.71)% and (28.39 ±5.28)% respectively,while in NSP cells that was (0. 26 ±0. 06)%, (2. 51 ±0. 17)%, ( 1.74 ±0. 38)%, ( 1.52 ±0. 41 )% and ( 3.37 ± 1.02) % respectively ( P < 0. 01 ). Conclusion The SP cells sorted from SMMC-7721 cell line may enrich tumor stem cells. Purified liver cancer stem cells may be obtained by screening SP cells using a variety of stem cell markers.%目的 分选肝癌细胞株SMMC-7721中的侧群(SP)细胞,并分析其干细胞标记的表达.方法 采用流式细胞荧光激活分选(FACS)技术将SMMC-7721细胞分为SP细胞和非侧群(NSP)细胞两个亚群,以实时荧光定量聚合酶链反应(real-time PCR)技术和流式细胞术对两个亚群细胞干细胞标记mRNA和蛋白表达进行分析.结果 SMMC-7721细胞株中分选出的SP细胞比例为(9.2±0.2)%.SP细胞ABCG2、CD133、Oct4、Sox2和NANOG等干细胞标记mRNA的表达水平分别是NSP细胞的7.132倍、4.985倍、8.642倍、5.095倍和5.164倍,差异均有统计学意义(P<0.01);ABCG2、CD133、Oct4、Sox2和NANOG蛋白在

  16. What is a Sorting Function?

    DEFF Research Database (Denmark)

    Henglein, Fritz

    2009-01-01

    What is a sorting function—not a sorting function for a given ordering relation, but a sorting function with nothing given? Formulating four basic properties of sorting algorithms as defining requirements, we arrive at intrinsic notions of sorting and stable sorting: A function is a sorting...

  17. Yeast carboxypeptidase Y requires glycosylation for efficient intracellular transport, but not for vacuolar sorting, in vivo stability, or activity

    DEFF Research Database (Denmark)

    Winther, Jakob R.; Stevens, T H; Kielland-Brandt, Morten

    1991-01-01

    and intracellular sorting, and the stabilities in vivo and in vitro were studied. It was found that carbohydrate was not important for accurate vacuolar targeting of CPY, but that the rate of transport of the unglycosylated CPY through the secretory pathway to the vacuole was reduced. Tunicamycin, which inhibits...

  18. New connections: Cell to cell HIV-1 transmission, resistance to broadly neutralizing antibodies, and an envelope sorting motif.

    Science.gov (United States)

    Smith, S Abigail; Derdeyn, Cynthia A

    2017-03-01

    HIV-1 infection from cell to cell may provide an efficient mode of viral spread in vivo and could therefore present a significant challenge for preventative or therapeutic strategies based on broadly neutralizing antibodies. Indeed, Li et al show that the potency and magnitude of multiple HIV-1 broadly neutralizing antibody classes are decreased during cell to cell infection in a context dependent manner. A functional motif in gp41 appears to contribute to this differential susceptibility by modulating exposure of neutralization epitopes.

  19. Raman activated cell ejection for isolation of single cells.

    Science.gov (United States)

    Wang, Yun; Ji, Yuetong; Wharfe, Emma S; Meadows, Roger S; March, Peter; Goodacre, Royston; Xu, Jian; Huang, Wei E

    2013-11-19

    We have optimized a Raman microscope to obtain a single cell Raman spectrum (SCRS) with 0.1 s acquisition time. SCRS with such short acquisition time has sufficient discriminatory ability and spectral reproducibility to differentiate cells incorporated with (13)C and (15)N and to classify five different types of bacteria isolated from the oral cavity. We also developed Raman activated cell ejection (RACE) that is assisted by laser induced forward transfer (LIFT). We have shown, for the first time, that the single cells of interest can be identified and then accurately isolated from complex microbial communities based on their SCRS. This approach can be used to sort single cells of target traits from complex samples (e.g., biofilms, soils, sludge, tissues).

  20. Parallel sorting algorithms

    CERN Document Server

    Akl, Selim G

    1985-01-01

    Parallel Sorting Algorithms explains how to use parallel algorithms to sort a sequence of items on a variety of parallel computers. The book reviews the sorting problem, the parallel models of computation, parallel algorithms, and the lower bounds on the parallel sorting problems. The text also presents twenty different algorithms, such as linear arrays, mesh-connected computers, cube-connected computers. Another example where algorithm can be applied is on the shared-memory SIMD (single instruction stream multiple data stream) computers in which the whole sequence to be sorted can fit in the

  1. Femtosecond laser fabricated microfluorescence-activated cell sorter for single cell recovery

    Science.gov (United States)

    Bragheri, F.; Paiè, P.; Nava, G.; Yang, T.; Minzioni, P.; Martinez Vazquez, R.; Bellini, N.; Ramponi, R.; Cristiani, I.; Osellame, R.

    2014-03-01

    Manipulation, sorting and recovering of specific live cells from samples containing less than a few thousand cells is becoming a major hurdle in rare cell exploration such as stem cell research or cell based diagnostics. Moreover the possibility of recovering single specific cells for culturing and further analysis would be of great impact in many biological fields ranging from regenerative medicine to cancer therapy. In recent years considerable effort has been devoted to the development of integrated and low-cost optofluidic devices able to handle single cells, which usually rely on microfluidic circuits that guarantee a controlled flow of the cells. Among the different microfabrication technologies, femtosecond laser micromachining (FLM) is ideally suited for this purpose as it provides the integration of both microfluidic and optical functions on the same glass chip leading to monolithic, robust and portable devices. Here a new optofluidic device is presented, which is capable of sorting and recovering of single cells, through optical forces, on the basis of their fluorescence and. Both fluorescence detection and single cell sorting functions are integrated in the microfluidic chip by FLM. The device, which is specifically designed to operate with a limited amount of cells but with a very high selectivity, is fabricated by a two-step process that includes femtosecond laser irradiation followed by chemical etching. The capability of the device to act as a micro fluorescence-activated cell sorter has been tested on polystyrene beads and on tumor cells and the results on the single live cell recovery are reported.

  2. DNA methylation profiling of sorted cells from myelofibrosis patients reveals aberrant epigenetic regulation of immune pathways and identifies early MPN driver genes

    DEFF Research Database (Denmark)

    Nielsen, H. M.; Andersen, C. L.; Kristensen, L. S.

    2015-01-01

    , PV) toadvancedMF. Multiple studies report frequent mutations in epigenetic regulators. However, the association to epigenetic changes and the role of epigenetic aberrations in different cell populations is still unknown. Aims: We therefore performed DNA methylation profiling of sorted cells from MF...... and PV patients. Results: The number of differentially methylated CpG sites between MF cells and the respective counterparts from healthy donors differed extensively among the three cell populations analyzed. In MF CD34+ cells 1628 CpG sites were differentially methylated compared to normal CD34+ cells......Background: Primary myelofibrosis (PMF) belongs to the heterogeneous group of chronic myeloproliferative neoplasms (MPN) together with essential thrombocytosis (ET) and polycythemia vera (PV). It has been suggested that these neoplasms represent a biological continuum from early cancer stage (ET...

  3. Development of a cell sorting procedure to increase the sensitivity of detection of protein-protein interactions in plant protoplasts.

    Science.gov (United States)

    Zhang, Xin; Wong, Sek Man

    2011-05-01

    To visualize subcellular localization of viral proteins and interactions between viral proteins and host proteins in vivo, transfection of plasmids into protoplasts to over-express transiently fusion proteins with a fluorescent tag is a common method. However, due to the low efficiency (0.1-3.0%) of plasmid transfection into protoplasts, it is difficult to identify protoplasts that emit fluorescence using confocal microscopy. A flow cytometry sorting protocol was developed for separating kenaf protoplasts that emit yellow fluorescence. The sorted protoplasts showed strong fluorescence and the protoplasts were intact. This will improve the use of confocal microscopy for studying subcellular localization and protein interactions in protoplasts isolated from plants with low transfection efficiency.

  4. Sorting a distribution theory

    CERN Document Server

    Mahmoud, Hosam M

    2011-01-01

    A cutting-edge look at the emerging distributional theory of sorting Research on distributions associated with sorting algorithms has grown dramatically over the last few decades, spawning many exact and limiting distributions of complexity measures for many sorting algorithms. Yet much of this information has been scattered in disparate and highly specialized sources throughout the literature. In Sorting: A Distribution Theory, leading authority Hosam Mahmoud compiles, consolidates, and clarifies the large volume of available research, providing a much-needed, comprehensive treatment of the

  5. Designing sorting networks

    CERN Document Server

    Baddar, Sherenaz W Al-Haj

    2012-01-01

    Designing Sorting Networks: A New Paradigm provides an in-depth guide to maximizing the efficiency of sorting networks, and uses 0/1 cases, partially ordered sets and Haase diagrams to closely analyze their behavior in an easy, intuitive manner. This book also outlines new ideas and techniques for designing faster sorting networks using Sortnet, and illustrates how these techniques were used to design faster 12-key and 18-key sorting networks through a series of case studies. Finally, it examines and explains the mysterious behavior exhibited by the fastest-known 9-step 16-key network. Designi

  6. Sorting it out: regulation of exosome loading.

    Science.gov (United States)

    Villarroya-Beltri, Carolina; Baixauli, Francesc; Gutiérrez-Vázquez, Cristina; Sánchez-Madrid, Francisco; Mittelbrunn, María

    2014-10-01

    Extracellular vesicles (EVs), a term that includes both exosomes of endocytic origin and vesicles derived from plasma membranes, are continuously secreted by cells to the extracellular environment, and represent a novel vehicle for cell-cell communication. Exosomes contain specific repertoires of proteins and RNAs, indicating the existence of mechanisms that control the sorting of molecules into them. Although the molecular mechanisms that regulate the loading of proteins into exosomes have been studied for years, the sorting of RNA has been elusive until recently. Here we review the molecular mechanisms that control the sorting of molecules into exosomes, with special attention to the sorting of RNA. We also discuss how the cellular context affects the composition of exosomes, and thus the outcome of the communication between the exosome-producer and recipient cells, with particular focus on the communication between tumor cells and with cells of the tumor microenvironment.

  7. Multiparametric Flow Cytometry and Cell Sorting for the Assessment of Viable, Injured, and Dead Bifidobacterium Cells during Bile Salt Stress

    OpenAIRE

    2002-01-01

    Using a flow cytometry-based approach, we assessed the viability of Bifidobacterium lactis DSM 10140 and Bifidobacterium adolescentis DSM 20083 during exposure to bile salt stress. Carboxyfluorescein diacetate (cFDA), propidium iodide (PI), and oxonol [DiBAC4(3)] were used to monitor esterase activity, membrane integrity, and membrane potential, respectively, as indicators of bacterial viability. Single staining with these probes rapidly and noticeably reflected the behavior of the two strain...

  8. Automated Sorting of Transuranic Waste

    Energy Technology Data Exchange (ETDEWEB)

    Shurtliff, Rodney Marvin

    2001-03-01

    The HANDSS-55 Transuranic Waste Sorting Module is designed to sort out items found in 55-gallon drums of waste as determined by an operator. Innovative imaging techniques coupled with fast linear motor-based motion systems and a flexible end-effector system allow the operator to remove items from the waste stream by a touch of the finger. When all desired items are removed from the waste stream, the remaining objects are automatically moved to a repackaging port for removal from the glovebox/cell. The Transuranic Waste Sorting Module consists of 1) a high accuracy XYZ Stereo Measurement and Imaging system, 2) a vibrating/tilting sorting table, 3) an XY Deployment System, 4) a ZR Deployment System, 5) several user-selectable end-effectors, 6) a waste bag opening system, 7) control and instrumentation, 8) a noncompliant waste load-out area, and 9) a Human/Machine Interface (HMI). The system is modular in design to accommodate database management tools, additional load-out ports, and other enhancements. Manually sorting the contents of a 55-gallon drum takes about one day per drum. The HANDSS-55 Waste Sorting Module is designed to significantly increase the throughput of this sorting process by automating those functions that are strenuous and tiresome for an operator to perform. The Waste Sorting Module uses the inherent ability of an operator to identify the items that need to be segregated from the waste stream and then, under computer control, picks that item out of the waste and deposits it in the appropriate location. The operator identifies the object by locating the visual image on a large color display and touches the image on the display with his finger. The computer then determines the location of the object, and performing a highspeed image analysis determines its size and orientation, so that a robotic gripper can be deployed to pick it up. Following operator verification by voice or function key, the object is deposited into a specified location.

  9. Sorting out Downside Beta

    NARCIS (Netherlands)

    G.T. Post (Thierry); P. van Vliet (Pim); S.D. Lansdorp (Simon)

    2009-01-01

    textabstractDownside risk, when properly defined and estimated, helps to explain the cross-section of US stock returns. Sorting stocks by a proper estimate of downside market beta leads to a substantially larger cross-sectional spread in average returns than sorting on regular market beta. This

  10. Isolation and Tumorigenicity of Side Population Cells Sorted from Hepatocellular Carcinoma Bel-7402 Cell Line%肝癌细胞株Bel-7402中侧群细胞的分离和致瘤能力

    Institute of Scientific and Technical Information of China (English)

    陈伟; 姜楠; 张彤; 李华; 张琪; 陈规划; 曾宪成

    2012-01-01

    [Objective] To study the biological characteristics of side population (SP) cells sorted from hepatocellular carcinoma Bel-7402 cell line. [Methods] Fluorescence-activated cell sorter (FACS) was used to sort SP cells and non-SP (NSP) cells from Bel-7402 cell line. Groups were: SP group and NSP group. The colony formation and proliferation ability were compared between SP cells and NSP cells by plate colony and growth curve assay. ATP-binding cassette superfamily G member 2 (ABCG2) and CD133 mRNA levels in SP and NSP cells were examined by RT-PCR. The apoptosis was analyzed by flow cytometry. The sphere rate was counted for SP cells and NSP cells in serum-free medium. The oncogenicity of the SP and NSP cells were analyzed by tumor formation in nonobese diabeti- c/severe combined immune- deficient (NOD/SCID) mice. [Results] (2.4 ?.0) % SP cells were sorted from the Bel-7402 cells by FACS. Growth curve assay showed that the proliferation ability of SP cells was higher than NSP cells (P 0.05, P < 0.05,and P < 0.05). [Conclusion] The SP cells sorted from Bel-7402 cell line may enrich tumor stem cells.%[目的]分选肝癌Bel-7402细胞中的侧群(SP)细胞并分析其生物学特性.[方法]采用流式细胞荧光激活分选(FACS)技术将Bel-7402细胞分为SP细胞和非侧群(NSP)细胞2亚群.实验分组:SP组与NSP组.细胞生长曲线法和平板克隆法比较SP细胞和NSP细胞的增殖能力和克隆形成能力;RT-PCR法检测SP细胞和NSP细胞的CD133、三磷酸腺苷结合盒转动蛋白G2(ABCG2)表达;流式细胞术分析细胞凋亡率;无血清培养法检测SP细胞及NSP细胞成球率;非肥胖糖尿病/严重联合免疫缺陷(NOD/SCID)小鼠体内成瘤实验比较SP细胞和NSP细胞的致瘤性.[结果]Bel-7402细胞中分选出的SP细胞比例为(2.4±0.0)%.生长曲线表明SP细胞的增殖速度快于NSP细胞(P<0.05).SP、NSP细胞的克隆形成率分别为(20.5±2.6)%、(5.1±0.9)%,两者差异明显(P<0.05).SP

  11. Cloning and subcellular location of an arabidopsis receptor-like protein that shares common features with protein-sorting receptors of eukaryotic cells

    Energy Technology Data Exchange (ETDEWEB)

    Ahmed, S.U.; Bar-Peled, M.; Raikhel, N.V. [Michigan State Univ., East Lansing, MI (United States)

    1997-05-01

    Many receptors involved in clathrin-mediated protein transport through the endocytic and secretary pathways of yeast and animal cells share common features. They are all type I integral membrane proteins containing cysteine-rich lumenal domains and cytoplasmic tails with tyrosine-containing sorting signals. The cysteine-rich domains are thought to be involved in ligand binding, whereas the cytoplasmic tyrosine motifs interact with clathrin-associated adaptor proteins during protein sorting along these pathways. in addition, tyrosine-containing signals are required for the retention and recycling of some of these membrane proteins to the trans-Golgi network. Here we report the characterization of an approximately 80-kD epidermal growth factor receptor-like type I integral membrane protein containing all of these functional motifs from Arabidopsis thaliana (called AtELP for A. thaliana Epidermal growth factor receptor-Like Protein). Biochemical analysis indicates that AtELP is a membrane protein found at high levels in the roots of both monocots and dicots. Subcellular fractionation studies indicate that the AtELP protein is present in two membrane fractions corresponding to a novel, undefined compartment and a fraction enriched in vesicles containing clathrin and its associated adaptor proteins. AtELP may therefore serve as a marker for compartments involved in intracellular protein trafficking in the plant cell. 87 refs., 7 figs.

  12. Activity of the C-terminal-dependent vacuolar sorting signal of horseradish peroxidase C1a is enhanced by its secondary structure.

    Science.gov (United States)

    Matsui, Takeshi; Tabayashi, Ayako; Iwano, Megumi; Shinmyo, Atsuhiko; Kato, Ko; Nakayama, Hideki

    2011-02-01

    Plant class III peroxidase (PRX) catalyzes the oxidation and oxidative polymerization of a variety of phenolic compounds while reducing hydrogen peroxide. PRX proteins are classified into apoplast type and vacuole type based on the absence or the presence of C-terminal propeptides, which probably function as vacuolar sorting signals (VSSs). In this study, in order to improve our understanding of vacuole-type PRX, we analyzed regulatory mechanisms of vacuolar sorting of a model vacuole-type PRX, the C1a isozyme of horseradish (Armoracia rusticana) (HRP C1a). Using cultured transgenic tobacco cells and protoplasts derived from horseradish leaves, we characterized HRP C1a's VSS, which is a 15 amino acid C-terminal propeptide (C15). We found that the C-terminal hexapeptide of C15 (C6), which is well conserved among vacuole-type PRX proteins, forms the core of the C-terminal-dependent VSS. We also found that the function of C6 is enhanced by the remaining N-terminal part of C15 which probably folds into an amphiphilic α-helix.

  13. Old world arenaviruses enter the host cell via the multivesicular body and depend on the endosomal sorting complex required for transport.

    Directory of Open Access Journals (Sweden)

    Giulia Pasqual

    2011-09-01

    Full Text Available The highly pathogenic Old World arenavirus Lassa virus (LASV and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV use α-dystroglycan as a cellular receptor and enter the host cell by an unusual endocytotic pathway independent of clathrin, caveolin, dynamin, and actin. Upon internalization, the viruses are delivered to acidified endosomes in a Rab5-independent manner bypassing classical routes of incoming vesicular trafficking. Here we sought to identify cellular factors involved in the unusual and largely unknown entry pathway of LASV and LCMV. Cell entry of LASV and LCMV required microtubular transport to late endosomes, consistent with the low fusion pH of the viral envelope glycoproteins. Productive infection with recombinant LCMV expressing LASV envelope glycoprotein (rLCMV-LASVGP and LCMV depended on phosphatidyl inositol 3-kinase (PI3K as well as lysobisphosphatidic acid (LBPA, an unusual phospholipid that is involved in the formation of intraluminal vesicles (ILV of the multivesicular body (MVB of the late endosome. We provide evidence for a role of the endosomal sorting complex required for transport (ESCRT in LASV and LCMV cell entry, in particular the ESCRT components Hrs, Tsg101, Vps22, and Vps24, as well as the ESCRT-associated ATPase Vps4 involved in fission of ILV. Productive infection with rLCMV-LASVGP and LCMV also critically depended on the ESCRT-associated protein Alix, which is implicated in membrane dynamics of the MVB/late endosomes. Our study identifies crucial cellular factors implicated in Old World arenavirus cell entry and indicates that LASV and LCMV invade the host cell passing via the MVB/late endosome. Our data further suggest that the virus-receptor complexes undergo sorting into ILV of the MVB mediated by the ESCRT, possibly using a pathway that may be linked to the cellular trafficking and degradation of the cellular receptor.

  14. Old world arenaviruses enter the host cell via the multivesicular body and depend on the endosomal sorting complex required for transport.

    Science.gov (United States)

    Pasqual, Giulia; Rojek, Jillian M; Masin, Mark; Chatton, Jean-Yves; Kunz, Stefan

    2011-09-01

    The highly pathogenic Old World arenavirus Lassa virus (LASV) and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) use α-dystroglycan as a cellular receptor and enter the host cell by an unusual endocytotic pathway independent of clathrin, caveolin, dynamin, and actin. Upon internalization, the viruses are delivered to acidified endosomes in a Rab5-independent manner bypassing classical routes of incoming vesicular trafficking. Here we sought to identify cellular factors involved in the unusual and largely unknown entry pathway of LASV and LCMV. Cell entry of LASV and LCMV required microtubular transport to late endosomes, consistent with the low fusion pH of the viral envelope glycoproteins. Productive infection with recombinant LCMV expressing LASV envelope glycoprotein (rLCMV-LASVGP) and LCMV depended on phosphatidyl inositol 3-kinase (PI3K) as well as lysobisphosphatidic acid (LBPA), an unusual phospholipid that is involved in the formation of intraluminal vesicles (ILV) of the multivesicular body (MVB) of the late endosome. We provide evidence for a role of the endosomal sorting complex required for transport (ESCRT) in LASV and LCMV cell entry, in particular the ESCRT components Hrs, Tsg101, Vps22, and Vps24, as well as the ESCRT-associated ATPase Vps4 involved in fission of ILV. Productive infection with rLCMV-LASVGP and LCMV also critically depended on the ESCRT-associated protein Alix, which is implicated in membrane dynamics of the MVB/late endosomes. Our study identifies crucial cellular factors implicated in Old World arenavirus cell entry and indicates that LASV and LCMV invade the host cell passing via the MVB/late endosome. Our data further suggest that the virus-receptor complexes undergo sorting into ILV of the MVB mediated by the ESCRT, possibly using a pathway that may be linked to the cellular trafficking and degradation of the cellular receptor.

  15. Increased basolateral sorting of carcinoembryonic antigen in a polarized colon carcinoma cell line after cholesterol depletion-Implications for treatment of inflammatory bowel disease

    Institute of Scientific and Technical Information of China (English)

    Robert Ehehalt; Markus Krautter; Martin Zorn; Richard Sparla; Joachim Fūllekrug; Hasan Kulaksiz; Wolfgang Stremmel

    2008-01-01

    AIM:To investigate a possible increase of basolateral expression of carcinoembryonic antigen(CEA)by interfering with the apical transport machinery,we studied the effect of cholesterol depletion on CEA sorting and secretion.METHODS:Cholesterol depletion was performed in polarized Caco-2 cells using Iovastatin and methyl-βcyclodextrin.RESULTS:We show that CEA is predominantly expressed and secreted at the apical surface.Reduction of the cholesterol level of the cell by 40%-50% with Iovastatin and methyl-β-cyclodextrin led to a significant change of the apical-to-basolateral transport ratio towards the basolateral membrane.CONCLUSION:As basolateral expression of CEA has been suggested to have anti-inflamatory properties,Cholesterol depletion of enterocytes might be a potential approach to influence the course of inflammatory bowel disease.

  16. Card Sorts, State Tests, and Meaningful Mathematics

    Science.gov (United States)

    Chauvot, Jennifer B.; Benson, Sharon L. D.

    2008-01-01

    This article shares card-sorting activities that involve state-mandated test items to use with prospective and practicing mathematics teachers to teach about accountability measures while exploring reform-minded mathematics instruction recommendations. (Contains 2 figures.)

  17. Layers in sorting practices: Sorting out patients with potential cancer

    DEFF Research Database (Denmark)

    Møller, Naja Holten; Bjørn, Pernille

    2011-01-01

    mechanism, but is handled by informal sorting mechanisms. We identify two informal sorting mechanisms with large impact on the sorting practices, namely subtle categorizing and collective remembering. These informal sorting mechanisms have implications for the design of electronic booking systems because...

  18. Identification of HLA-B27-restricted peptides in reactive arthritis and other spondyloarthropathies: computer algorithms and fluorescent activated cell sorting analysis as tools for hunting of HLA-B27-restricted chlamydial and autologous crossreactive peptides involved in reactive arthritis and ankylosing spondylitis.

    Science.gov (United States)

    Kuon, Wolfgang; Sieper, Joachim

    2003-08-01

    The illustrated clinical and experimental results demonstrate the strong relationship between the MHC class I antigen HLA-B27 and synovial CD8+ T cells with specificity for bacterial and possible self-antigen in SpA. These new aspects obtained in recent experimental and clinical studies might also provide clues to the pathomechanisms of joint inflammation in SpA. In particular, the newly developed techniques will be of great relevance in the near future. New and more precise bioalgorithms reflecting new insights in the biology and biochemistry of proteins as recently presented [98, 99] can be helpful (e.g., a program with an improved prediction of the features of immunoproteasomes). Intracellular and secreted cytokine staining by FACScan allows examination of a great number of cells expressing certain antigens in response to certain stimuli. The analysis of T-cell responses with tetramer/peptide complexes can be useful to screen tissue sections for TCR, recognizing foreign or self-derived epitopes on those complexes loaded with selected (e.g., bacterial) peptides. Identification of arthritogenic peptides and a further understanding of the immunology of the pathomechanisms in SpA might open ways to design new peptide vaccines to prevent inflammation, autoimmunity, and other diseases by early intervention [100].

  19. LazySorted: A Lazily, Partially Sorted Python List

    Directory of Open Access Journals (Sweden)

    Naftali Harris

    2015-06-01

    Full Text Available LazySorted is a Python C extension implementing a partially and lazily sorted list data structure. It solves a common problem faced by programmers, in which they need just part of a sorted list, like its middle element (the median, but sort the entire list to get it. LazySorted presents them with the abstraction that they are working with a fully sorted list, while actually only sorting the list partially with quicksort partitions to return the requested sub-elements. This enables programmers to use naive "sort first" algorithms but nonetheless attain linear run-times when possible. LazySorted may serve as a drop-in replacement for the built-in sorted function in most cases, and can sometimes achieve run-times more than 7 times faster.

  20. Exosome and Exosomal MicroRNA:Trafficking, Sorting, and Function

    Institute of Scientific and Technical Information of China (English)

    Jian Zhang; Sha Li; Lu Li; Meng Li; Chongye Guo; Jun Yao; Shuangli Mi

    2015-01-01

    Exosomes are 40–100 nm nano-sized vesicles that are released from many cell types into the extracellular space. Such vesicles are widely distributed in various body fluids. Recently, mRNAs and microRNAs (miRNAs) have been identified in exosomes, which can be taken up by neighboring or distant cells and subsequently modulate recipient cells. This suggests an active sort-ing mechanism of exosomal miRNAs, since the miRNA profiles of exosomes may differ from those of the parent cells. Exosomal miRNAs play an important role in disease progression, and can stimu-late angiogenesis and facilitate metastasis in cancers. In this review, we will introduce the origin and the trafficking of exosomes between cells, display current research on the sorting mechanism of exo-somal miRNAs, and briefly describe how exosomes and their miRNAs function in recipient cells. Finally, we will discuss the potential applications of these miRNA-containing vesicles in clinical settings.

  1. Sorting Plastic Waste in Hydrocyclone

    Directory of Open Access Journals (Sweden)

    Ernestas Šutinys

    2011-02-01

    Full Text Available The article presents material about sorting plastic waste in hydrocyclone. The tests on sorting plastic waste were carried out. Also, the findings received from the performed experiment on the technology of sorting plastic waste are interpreted applying an experimental model of the equipment used for sorting plastics of different density.Article in Lithuanian

  2. Ready, steady, SORT!

    CERN Multimedia

    Laëtitia Pedroso

    2010-01-01

    The selective or ecological sorting of waste is already second nature to many of us and concerns us all. As the GS Department's new awareness-raising campaign reminds us, everything we do to sort waste contributes to preserving the environment.    Placemats printed on recycled paper using vegetable-based ink will soon be distributed in Restaurant No.1.   Environmental protection is never far from the headlines, and CERN has a responsibility to ensure that the 3000 tonnes and more of waste it produces every year are correctly and selectively sorted. Materials can be given a second life through recycling and re-use, thereby avoiding pollution from landfill sites and incineration plants and saving on processing costs. The GS Department is launching a new poster campaign designed to raise awareness of the importance of waste sorting and recycling. "After conducting a survey to find out whether members of the personnel were prepared to make an effort to sort a...

  3. The assessment of CD146-based cell sorting and telomere length analysis for establishing the identity of mesenchymal stem cells in human umbilical cord [v2; ref status: indexed, http://f1000r.es/48d

    Directory of Open Access Journals (Sweden)

    Dimitrios Kouroupis

    2014-08-01

    Full Text Available Adult stem cells are characterised by longer telomeres compared to mature cells from the same tissue. In this study, candidate CD146+ umbilical cord (UC mesenchymal stem cells (MSCs were purified by cell sorting from UC tissue digests and their telomere lengths were measured in comparison to donor-matched CD146-negative fraction.   UC tissue fragments were enzymatically treated with collagenase and the cells were used for cell sorting, colony-forming fibroblast (CFU-F assay or for long-term MSC cultivation. Telomere lengths were measured by qPCR in both culture-expanded MSCs and candidate native UC MSCs. Immunohistochemistry was undertaken to study the topography of CD146+ cells.   Culture-expanded UC MSCs had a stable expression of CD73, CD90 and CD105, whereas CD146 declined in later passages which correlated with the shortening of telomeres in the same cultures. In five out of seven donors, telomeres in candidate native UC MSCs (CD45-CD235α-CD31-CD146+ were longer compared to donor-matched CD146- population (CD45-CD235α-CD31-CD146-. The frequency of CD45-CD235α-CD31-CD146+ cells measured by flow cytometry was ~1000-fold above that of CFU-Fs (means 10.4% and 0.01%, respectively. CD146+ cells were also abundant in situ having a broad topography including high levels of positivity in muscle areas in addition to vessels.   Although qPCR-based telomere length analysis in sorted populations could be limited in its sensitivity, very high frequency of CD146+ cells in UC tissue suggests that CD146 expression alone is unlikely to be sufficient to identify and purify native MSCs from the UC tissue.

  4. Relationship of severity of subacute ruminal acidosis to rumen fermentation, chewing activities, sorting behavior, and milk production in lactating dairy cows fed a high-grain diet.

    Science.gov (United States)

    Gao, X; Oba, M

    2014-05-01

    The objectives of the current study were to evaluate the variation in severity of subacute ruminal acidosis (SARA) among lactating dairy cows fed a high-grain diet and to determine factors characterizing animals that are tolerant to high-grain diets. Sixteen ruminally cannulated late-lactating dairy cows (days in milk=282 ± 33.8; body weight=601 ± 75.9 kg) were fed a high-grain diet consisting of 35% forage and 65% concentrate mix. After 17 d of diet adaptation, chewing activities were monitored for a 24-h period and ruminal pH was measured every 30s for 72 h. Acidosis index, defined as the severity of SARA (area of pH acidosis index ranged from 0.0 to 10.9 pH · min/kg of DMI. Six cows with the lowest acidosis index (0.04 ± 0.61 pH · min/kg) and 4 with the highest acidosis index (7.67 ± 0.75 pH · min/kg) were classified as animals that were tolerant and susceptible to the high-grain diet, respectively. Total volatile fatty acid concentration and volatile fatty acid profile were not different between the groups. Susceptible animals sorted against long particles, whereas tolerant animals did not (sorting index=87.6 vs. 97.9, respectively). However, the tolerant cows had shorter total chewing time (35.8 vs. 45.1 min/kg of DMI). In addition, although DMI, milk yield, and milk component yields did not differ between the groups, milk urea nitrogen concentration was higher for tolerant cows compared with susceptible cows (12.8 vs. 8.6 mg/dL), which is possibly attributed to less organic matter fermentation in the rumen of tolerant cows. These results suggest that a substantial variation exists in the severity of SARA among lactating dairy cows fed the same high-grain diet, and that cows tolerant to the high-grain diet might be characterized by less sorting behavior but less chewing time, and higher milk urea nitrogen concentration. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  5. A non-destructive culturing and cell sorting method for cardiomyocytes and neurons using a double alginate layer.

    Directory of Open Access Journals (Sweden)

    Hideyuki Terazono

    Full Text Available A non-destructive method of collecting cultured cells after identifying their in situ functional characteristics is proposed. In this method, cells are cultivated on an alginate layer in a culture dish and released by spot application of a calcium chelate buffer that locally melts the alginate layer and enables the collection of cultured cells at the single-cell level. Primary hippocampal neurons, beating human embryonic stem (hES cell-derived cardiomyocytes, and beating hES cell-derived cardiomyocyte clusters cultivated on an alginate layer were successfully released and collected with a micropipette. The collected cells were recultured while maintaining their physiological function, including beating, and elongated neurites. These results suggest that the proposed method may eventually facilitate the transplantation of ES- or iPS-derived cardiomyocytes and neurons differentiated in culture.

  6. Sorting by Recursive Partitioning,

    Science.gov (United States)

    1983-12-01

    asymptotic time-complexity. This paper has the following main parts: First, a Pidgin -Algol version of the algorithm is presented and we discuss the main...those sorted subsets e) end "UsingBin*; end "AdaptSorting. 4 "Figure 1: A condensed Pidgin -Algol version of Adaptsort eiFor some conditions that we will...algorithm which have to be completed in either linear or constant times (these required critical times appear as comments in the Pidgin -Algol version

  7. Wage Sorting Trends

    DEFF Research Database (Denmark)

    Bagger, Jesper; Vejlin, Rune Majlund; Sørensen, Kenneth Lykke

    Using a population-wide Danish Matched Employer-Employee panel from 1980-2006, we document a strong trend towards more positive assortative wage sorting. The correlation between worker and firm fixed effects estimated from a log wage regression increases from -0.07 in 1981 to .14 in 2001. The non......Using a population-wide Danish Matched Employer-Employee panel from 1980-2006, we document a strong trend towards more positive assortative wage sorting. The correlation between worker and firm fixed effects estimated from a log wage regression increases from -0.07 in 1981 to .14 in 2001...

  8. The Proper Criteria for Identification and Sorting of Very Small Embryonic-Like Stem Cells, and Some Nomenclature Issues

    Science.gov (United States)

    Suszynska, Malwina; Zuba-Surma, Ewa K.; Maj, Magdalena; Mierzejewska, Kasia; Ratajczak, Janina; Kucia, Magda

    2014-01-01

    Evidence has accumulated that both murine and human adult tissues contain early-development stem cells with a broader differentiation potential than other adult monopotent stem cells. These cells, being pluripotent or multipotent, exist at different levels of specification and most likely represent overlapping populations of cells that, depending on the isolation strategy, ex vivo expansion protocol, and markers employed for their identification, have been given different names. In this review, we will discuss a population of very small embryonic-like stem cells (VSELs) in the context of other stem cells that express pluripotent/multipotent markers isolated from adult tissues as well as review the most current, validated working criteria on how to properly identify and isolate these very rare cells. VSELs have been successfully purified in several laboratories; however, a few have failed to isolate them, which has raised some unnecessary controversy in the field. Therefore, in this short review, we will address the most important reasons that some investigators have experienced problems in isolating these very rare cells and discuss some still unresolved challenges which should be overcome before these cells can be widely employed in the clinic. PMID:24299281

  9. Alternative fluorescent labeling strategies for characterizing gram-positive pathogenic bacteria: Flow cytometry supported counting, sorting, and proteome analysis of Staphylococcus aureus retrieved from infected host cells.

    Science.gov (United States)

    Hildebrandt, Petra; Surmann, Kristin; Salazar, Manuela Gesell; Normann, Nicole; Völker, Uwe; Schmidt, Frank

    2016-10-01

    Staphylococcus aureus is a Gram-positive opportunistic pathogen that is able to cause a broad range of infectious diseases in humans. Furthermore, S. aureus is able to survive inside nonprofessional phagocytic host cell which serve as a niche for the pathogen to hide from the immune system and antibiotics therapies. Modern OMICs technologies provide valuable tools to investigate host-pathogen interactions upon internalization. However, these experiments are often hampered by limited capabilities to retrieve bacteria from such an experimental setting. Thus, the aim of this study was to develop a labeling strategy allowing fast detection and quantitation of S. aureus in cell lysates or infected cell lines by flow cytometry for subsequent proteome analyses. Therefore, S. aureus cells were labeled with the DNA stain SYTO(®) 9, or Vancomycin BODIPY(®) FL (VMB), a glycopeptide antibiotic binding to most Gram-positive bacteria which was conjugated to a fluorescent dye. Staining of S. aureus HG001 with SYTO 9 allowed counting of bacteria from pure cultures but not in cell lysates from infection experiments. In contrast, with VMB it was feasible to stain bacteria from pure cultures as well as from samples of infection experiments. VMB can also be applied for histocytochemistry analysis of formaldehyde fixed cell layers grown on coverslips. Proteome analyses of S. aureus labeled with VMB revealed that the labeling procedure provoked only minor changes on proteome level and allowed cell sorting and analysis of S. aureus from infection settings with sensitivity similar to continuous gfp expression. Furthermore, VMB labeling allowed precise counting of internalized bacteria and can be employed for downstream analyses, e.g., proteomics, of strains not easily amendable to genetic manipulation such as clinical isolates. © 2016 International Society for Advancement of Cytometry.

  10. Gender Differences in Sorting

    DEFF Research Database (Denmark)

    Merlino, Luca Paolo; Parrotta, Pierpaolo; Pozzoli, Dario

    In this paper, we investigate the sorting of workers in firms to understand gender gaps in labor market outcomes. Using Danish employer-employee matched data, we fiend strong evidence of glass ceilings in certain firms, especially after motherhood, preventing women from climbing the career ladder...

  11. Protein Sorting Prediction

    DEFF Research Database (Denmark)

    Nielsen, Henrik

    2017-01-01

    Many computational methods are available for predicting protein sorting in bacteria. When comparing them, it is important to know that they can be grouped into three fundamentally different approaches: signal-based, global-property-based and homology-based prediction. In this chapter, the strengths...

  12. Sorting and sustaining cooperation

    DEFF Research Database (Denmark)

    Vikander, Nick

    2013-01-01

    This paper looks at cooperation in teams where some people are selfish and others are conditional cooperators, and where lay-offs will occur at a fixed future date. I show that the best way to sustain cooperation prior to the lay-offs is often in a sorting equilibrium, where conditional cooperato...

  13. Det sorte USA

    DEFF Research Database (Denmark)

    Brøndal, Jørn

    Bogen gennemgår det sorte USAs historie fra 1776 til 2016, idet grundtemaet er spændingsforholdet mellem USAs grundlæggelsesidealer og den racemæssige praksis, et spændingsforhold som Gunnar Myrdal kaldte "det amerikanske dilemma." Bogen, der er opbygget som politisk, social og racemæssig historie...

  14. Online Sorted Range Reporting

    DEFF Research Database (Denmark)

    Brodal, Gerth Stølting; Fagerberg, Rolf; Greve, Mark

    2009-01-01

    We study the following one-dimensional range reporting problem: On an arrayA of n elements, support queries that given two indices i ≤ j and an integerk report the k smallest elements in the subarray A[i..j] in sorted order. We present a data structure in the RAM model supporting such queries in ...

  15. Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35★

    OpenAIRE

    Yan, Mingmin; Mao, Shanping; Dong, Huimin; Liu, Baohui; Zhang, Qian; PAN, GAOFENG; Fu, Zhiping

    2012-01-01

    PC12 cell injury was induced using 20 μM amyloid β-protein 25–35 to establish a model of Alzheimer's disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25–35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical...

  16. Visual ergonomics interventions in mail sorting facilities.

    Science.gov (United States)

    Hemphälä, H; Hansson, G-Å; Dahlqvist, C; Eklund, J

    2012-01-01

    This study was performed between 2004 and 2011 at mail sorting facilities in Sweden. During this time, different interventions were performed. The first was a lighting intervention that had a positive impact on the postal workers, especially those with eyestrain. A new lighting system also improved the illuminance and gave better light distribution. The second intervention involved new personal spectacles for the postal workers who needed them and this had a positive effect on eyestrain. The third intervention involved a specific type of sorting spectacles for the postal workers who already used progressive lenses privately. The reading distances that the postal workers had while sorting the mail was inverted to the distances in their regular progressive lenses. The new sorting spectacles had a positive effect on head postures and on muscular activity.

  17. Biological Characteristics of CD133+CD44+ Cancer Stem Cells Sorting from Laryngeal Carcinoma Cell Line TU177%喉癌TU177细胞系中CD133+CD44+肿瘤干细胞分选及特性分析

    Institute of Scientific and Technical Information of China (English)

    杨俊岭; 高伟; 王珏; 付荣; 陈波; 李伟艳; 温树信; 王斌全

    2016-01-01

    Objective :Magnetic activated cell sorting was used to separate CD133+CD44+ cancer cells from laryngeal car-cinoma TU177 cell line. Analysis the biological characteristics of these subpopulations .Methods :TU177 cells were subjected to magnetic activated cell sorting to obtain CD133+CD44+、CD133+CD44-、CD133-CD44+、CD133-CD44-cells. Evaluate the efficiency of magnetic separation by flow cytometry . Test cell proliferation,migration,invasion,adhesion,colony forming ability of the cells.Results: CD133+CD44+ cells show higher proliferation,migration,invasion,adhesion,clone ability than other group(P<0.0001).Conclusions:TU177 cells can be serparated by Magnetic activated cell sorting effectively. CD133 is more powerful than CD44.Our study may provide evidence for target treatment of laryngeal cancer.%目的:免疫磁珠分选喉癌TU177细胞系中的CD133+CD44+细胞,探讨CD133+CD44+细胞作为肿瘤干细胞的生物学特性。方法:培养喉癌TU177细胞,采用免疫磁珠分选技术分选CD133+CD44+、CD133+CD44-、CD133-CD44+、CD133-CD44-细胞,流式检测分选效率,检测各组细胞的增殖、侵袭、迁移、粘附、克隆形成能力。结果:CD133+CD44+细胞的增殖、迁移、侵袭、粘附、克隆能力均明显高于其他组,差异有统计学意义(P<0.0001)。结论:免疫磁珠技术能有效进行TU177细胞系的分选,CD133+CD44+细胞亚群具有强增殖、侵袭、迁移、粘附、克隆形成能力,具有肿瘤干细胞特征,CD133作为干细胞标志物,其干细胞特性强于CD44,可为喉癌的进一步靶向治疗提供依据。

  18. Sorting of ligand-activated epidermal growth factor receptor to lysosomes requires its actin-binding domain

    NARCIS (Netherlands)

    Stoorvogel, W; Kerstens, S; Fritzsche, I; den Hartigh, JC; Oud, R; van der Heyden, MAG; Henegouwen, PMPVE

    2004-01-01

    Ligand-induced down-regulation of the epidermal growth factor receptor (EGFR) comprises activation of two sequential transport steps. The first involves endocytic uptake by clathrin-coated vesicles, the second transfer of endocytosed EGFR from endosomes to lysosomes. Here we demonstrate that the sec

  19. Sorting nexin 1 loss results in D5 dopamine receptor dysfunction in human renal proximal tubule cells and hypertension in mice.

    Science.gov (United States)

    Villar, Van Anthony M; Jones, John Edward; Armando, Ines; Asico, Laureano D; Escano, Crisanto S; Lee, Hewang; Wang, Xiaoyan; Yang, Yu; Pascua-Crusan, Annabelle M; Palmes-Saloma, Cynthia P; Felder, Robin A; Jose, Pedro A

    2013-01-04

    The peripheral dopaminergic system plays a crucial role in blood pressure regulation through its actions on renal hemodynamics and epithelial ion transport. The dopamine D5 receptor (D(5)R) interacts with sorting nexin 1 (SNX1), a protein involved in receptor retrieval from the trans-Golgi network. In this report, we elucidated the spatial, temporal, and functional significance of this interaction in human renal proximal tubule cells and HEK293 cells stably expressing human D(5)R and in mice. Silencing of SNX1 expression via RNAi resulted in the failure of D(5)R to internalize and bind GTP, blunting of the agonist-induced increase in cAMP production and decrease in sodium transport, and up-regulation of angiotensin II receptor expression, of which expression was previously shown to be negatively regulated by D(5)R. Moreover, siRNA-mediated depletion of renal SNX1 in C57BL/6J and BALB/cJ mice resulted in increased blood pressure and blunted natriuretic response to agonist in salt-loaded BALB/cJ mice. These data demonstrate a crucial role for SNX1 in D(5)R trafficking and that SNX1 depletion results in D(5)R dysfunction and thus may represent a novel mechanism for the pathogenesis of essential hypertension.

  20. Selective isolation of ammonia-oxidizing bacteria from autotrophic nitrifying granules by applying cell-sorting and sub-culturing of microcolonies.

    Science.gov (United States)

    Fujitani, Hirotsugu; Kumagai, Asami; Ushiki, Norisuke; Momiuchi, Kengo; Tsuneda, Satoshi

    2015-01-01

    Nitrification is a key process in the biogeochemical nitrogen cycle and biological wastewater treatment that consists of two stepwise reactions, ammonia oxidation by ammonia-oxidizing bacteria (AOB) or archaea followed by nitrite oxidation by nitrite-oxidizing bacteria. One of the representatives of the AOB group is Nitrosomonas mobilis species. Although a few pure strains of this species have been isolated so far, approaches to their preservation in pure culture have not been established. Here, we report isolation of novel members of the N. mobilis species from autotrophic nitrifying granules used for ammonia-rich wastewater treatment. We developed an isolation method focusing on microcolonies formation of nitrifying bacteria. Two kinds of distinctive light scattering signatures in a cell-sorting system enabled to separate microcolonies from single cells and heterogeneous aggregates within granule samples. Inoculation of a pure microcolony into 96-well microtiter plates led to successful sub-culturing and increased probability of isolation. Obtained strain Ms1 is cultivated in the liquid culture with relatively high ammonia or nitrite concentration, not extremely slow growing. Considering environmental clones that were closely related to N. mobilis and detected in various environments, the availability of this novel strain would facilitate to reveal this member's ecophysiology in a variety of habitats.

  1. Selective isolation of ammonia-oxidizing bacteria from autotrophic nitrifying granules by applying cell-sorting and sub-culturing of microcolonies

    Directory of Open Access Journals (Sweden)

    Hirotsugu eFujitani

    2015-10-01

    Full Text Available Nitrification is a key process in the biogeochemical nitrogen cycle and biological wastewater treatment that consists of two stepwise reactions, ammonia oxidation by ammonia-oxidizing bacteria (AOB or archaea followed by nitrite oxidation by nitrite-oxidizing bacteria. One of the representative of the AOB group is Nitrosomonas mobilis species. Although a few pure strains of this species have been isolated so far, approaches to their preservation in pure culture have not been established. Here, we report isolation of novel members of the N. mobilis species from autotrophic nitrifying granules used for ammonia-rich wastewater treatment. We developed an isolation method focusing on microcolonies formation of nitrifying bacteria. Two kinds of distinctive light scattering signatures in a cell-sorting system enabled to separate microcolonies from single cells and heterogeneous aggregates within granule samples. Inoculation of a pure microcolony into 96-well microtiter plates led to successful sub-culturing and increased probability of isolation. Obtained strain Ms1 is cultivated in the liquid culture with relatively high ammonia or nitrite concentration, not extremely slow growing. Considering environmental clones that were closely related to N. mobilis and detected in various environments, the availability of this novel strain would facilitate to reveal this member’s ecophysiology in a variety of habitats.

  2. Immunological profiling of haemodialysis patients and young healthy individuals with implications for clinical regulatory T cell sorting.

    Science.gov (United States)

    Bergström, M; Joly, A-L; Seiron, P; Isringhausen, S; Modig, E; Fellström, B; Andersson, J; Berglund, D

    2015-05-01

    With the increasing interest in clinical trials with regulatory T cells (Tregs), immunological profiling of prospective target groups and standardized procedures for Treg isolation are needed. In this study, flow cytometry was used to assess peripheral blood lymphocyte profiles of young healthy individuals and patients undergoing haemodialysis treatment. Tregs obtained from the former may be used in haematopoietic stem cell transplantation and Tregs from the latter in the prevention of kidney transplant rejection. FOXP3 mRNA expression with accompanying isoform distribution was also assessed by the quantitative reverse transcriptase polymerase chain reaction. Flow-cytometric gating strategies were systematically analysed to optimize the isolation of Tregs. Our findings showed an overall similar immunological profile of both cohorts in spite of great differences in both age and health. Analysis of flow-cytometric gating techniques highlighted the importance of gating for both CD25high and CD127low expression in the isolation of FOXP3-positive cells. This study provides additional insight into the immunological profile of young healthy individuals and uraemic patients as well as in-depth analysis of flow-cytometric gating strategies for Treg isolation, supporting the development of Treg therapy using cells from healthy donors and uraemic patients.

  3. The effect of mechanical extension stimulation combined with epithelial cell sorting on outcomes of implanted tissue-engineered muscular urethras.

    Science.gov (United States)

    Fu, Qiang; Deng, Chen-Liang; Zhao, Ren-Yan; Wang, Ying; Cao, Yilin

    2014-01-01

    Urethral defects are common and frequent disorders and are difficult to treat. Simple natural or synthetic materials do not provide a satisfactory curative solution for long urethral defects, and urethroplasty with large areas of autologous tissues is limited and might interfere with wound healing. In this study, adipose-derived stem cells were used. These cells can be derived from a wide range of sources, have extensive expansion capability, and were combined with oral mucosal epithelial cells to solve the problem of finding seeding cell sources for producing the tissue-engineered urethras. We also used the synthetic biodegradable polymer poly-glycolic acid (PGA) as a scaffold material to overcome issues such as potential pathogen infections derived from natural materials (such as de-vascular stents or animal-derived collagen) and differing diameters. Furthermore, we used a bioreactor to construct a tissue-engineered epithelial-muscular lumen with a double-layer structure (the epithelial lining and the muscle layer). Through these steps, we used an epithelial-muscular lumen built in vitro to repair defects in a canine urethral defect model (1 cm). Canine urethral reconstruction was successfully achieved based on image analysis and histological techniques at different time points. This study provides a basis for the clinical application of tissue engineering of an epithelial-muscular lumen.

  4. K-sort: A new sorting algorithm that beats Heap sort for n <= 70 lakhs!

    CERN Document Server

    Sundararajan, Kiran Kumar; Chakraborty, Soubhik; Mahanti, N C

    2011-01-01

    Sundararajan and Chakraborty (2007) introduced a new version of Quick sort removing the interchanges. Khreisat (2007) found this algorithm to be competing well with some other versions of Quick sort. However, it uses an auxiliary array thereby increasing the space complexity. Here, we provide a second version of our new sort where we have removed the auxiliary array. This second improved version of the algorithm, which we call K-sort, is found to sort elements faster than Heap sort for an appreciably large array size (n <= 70,00,000) for uniform U[0, 1] inputs.

  5. Chip-based droplet sorting

    Energy Technology Data Exchange (ETDEWEB)

    Beer, Neil Reginald; Lee, Abraham; Hatch, Andrew

    2014-07-01

    A non-contact system for sorting monodisperse water-in-oil emulsion droplets in a microfluidic device based on the droplet's contents and their interaction with an applied electromagnetic field or by identification and sorting.

  6. Chip-based droplet sorting

    Science.gov (United States)

    Beer, Neil Reginald; Lee, Abraham; Hatch, Andrew

    2014-07-01

    A non-contact system for sorting monodisperse water-in-oil emulsion droplets in a microfluidic device based on the droplet's contents and their interaction with an applied electromagnetic field or by identification and sorting.

  7. Heideggers sorte arv

    DEFF Research Database (Denmark)

    Olesen, Søren Gosvig

    2015-01-01

    Martin Heidegger var antisemit, men er hans tænkning og intellektuelle arv det også? Søren Gosvig Olesen opsøger den store tyske tænkers arvinger og bindene fra 1938-48 i Heideggers efterladte ’Sorte hæfter’, hvor den lille mands meninger blander sig med en stor tænkers tanker......Martin Heidegger var antisemit, men er hans tænkning og intellektuelle arv det også? Søren Gosvig Olesen opsøger den store tyske tænkers arvinger og bindene fra 1938-48 i Heideggers efterladte ’Sorte hæfter’, hvor den lille mands meninger blander sig med en stor tænkers tanker...

  8. Sorting quantum systems efficiently

    Science.gov (United States)

    Ionicioiu, Radu

    2016-05-01

    Measuring the state of a quantum system is a fundamental process in quantum mechanics and plays an essential role in quantum information and quantum technologies. One method to measure a quantum observable is to sort the system in different spatial modes according to the measured value, followed by single-particle detectors on each mode. Examples of quantum sorters are polarizing beam-splitters (PBS) – which direct photons according to their polarization – and Stern-Gerlach devices. Here we propose a general scheme to sort a quantum system according to the value of any d-dimensional degree of freedom, such as spin, orbital angular momentum (OAM), wavelength etc. Our scheme is universal, works at the single-particle level and has a theoretical efficiency of 100%. As an application we design an efficient OAM sorter consisting of a single multi-path interferometer which is suitable for a photonic chip implementation.

  9. "Semi-straight sort of sex": class and gay community attachment explored within a framework of older homosexually active men.

    Science.gov (United States)

    Chapple, M J; Kippax, S; Smith, G

    1998-01-01

    Gay Community Attachment has proved a significant predictor of successful behavior change among gay-identifying men in response to HIV/AIDS. Related work at Macquarie University, Sydney, Australia, indicated that attachment to gay community is not a simple issue; rather, complex issues of sexual identity formation, the constraints of social inequality and localized sexual cultures inhibit the process of attachment and, therefore, successful HIV prevention. This paper discusses some of the findings from close-focus (qualitative) research on older homosexually active men which explore in depth the dynamic whereby these men attached themselves to gay community in terms of an analysis of class, generation, and the interplay with self-construction and masculinity.

  10. Microfluidic train station: highly robust and multiplexable sorting of droplets on electric rails.

    Science.gov (United States)

    Frenzel, Daniel; Merten, Christoph A

    2017-02-24

    Fluorescence-activated droplet sorting (FADS) has become a widely used technique for high-throughput screening applications. However, existing methods are very sensitive to fluctuating flow rates at the sorting junction, which can be caused by the pulsing effects of mechanical pumps, droplet aggregates or the accumulation of precipitates during lengthy biological screening applications. Furthermore, existing sorting devices allow only 2-way sorting. We present here a dielectrophoretic sorting system in which the droplets are sorted along multiple electrode pairs that run parallel to the channels. This enables highly reliable sorting (no errors were detected for more than 2000 sorting events) even when inverting the relative flow rates at a 2-way sorting junction from 80 : 20 to 20 : 80. Furthermore, our toolbox is scalable: we demonstrate on the example of a triple-colour sorting experiment with a total of four decoupled electrodes that multi-way sorting is feasible.

  11. The endosomal sorting complex required for transport (ESCRT) is required for the sensitivity of yeast cells to nickel ions in Saccharomyces cerevisiae.

    Science.gov (United States)

    Luo, Chong; Cao, Chunlei; Jiang, Linghuo

    2016-05-01

    Nickel is one of the toxic environment metal pollutants and is linked to various human diseases. In this study, through a functional genomics approach we have identified 16 nickel-sensitive and 22 nickel-tolerant diploid deletion mutants of budding yeast genes, many of which are novel players in the regulation of nickel homeostasis. The 16 nickel-sensitive mutants are of genes mainly involved in the protein folding, modification and destination and the cellular transport processes, while the 22 nickel-tolerant mutants are of genes encoding components of ESCRT complexes as well as protein factors involved in both the cell wall integrity maintenance and the vacuolar protein sorting process. In consistence with their phenotypes, most of these nickel-sensitive mutants show reduced intracellular nickel contents, while the majority of these nickel-tolerant mutants show elevated intracellular nickel contents, as compared to the wild type in response to nickel stress. Our data provides a basis for our understanding the regulation of nickel homeostasis and molecular mechanisms of nickel-induced human pathogenesis.

  12. Flow karyotyping and sorting of human chromosomes

    Energy Technology Data Exchange (ETDEWEB)

    Gray, J.W.; Lucas, J.; Peters, D.; Pinkel, D.; Trask, B.; van den Engh, G.; Van Dilla, M.A.

    1986-07-16

    Flow cytometry and sorting are becoming increasingly useful as tools for chromosome classfication and for the detection of numerical and structural chromosome aberrations. Chromosomes of a single type can be purified with these tools to facilitate gene mapping or production of chromosome specific recombinant DNA libraries. For analysis of chromosomes with flow cytometry, the chromosomes are extracted from mitotic cells, stained with one or more fluorescent dyes and classified one-by-one according to their dye content(s). Thus, the flow approach is fundamentally different than conventional karyotyping where chromosomes are classified within the context of a metaphase spread. Flow sorting allows purification of chromosomes that can be distinguished flow cytometrically. The authors describe the basic principles of flow cytometric chromosome classification i.e. flow karyotyping, and chromosome sorting and describe several applications. 30 refs., 8 figs.

  13. Spin-the-bottle Sort and Annealing Sort: Oblivious Sorting via Round-robin Random Comparisons

    CERN Document Server

    Goodrich, Michael T

    2010-01-01

    We study sorting algorithms based on randomized round-robin comparisons. Specifically, we study Spin-the-bottle sort, where comparisons are unrestricted, and Annealing sort, where comparisons are restricted to a distance bounded by a \\emph{temperature} parameter. Both algorithms are simple, randomized, data-oblivious sorting algorithms, which are useful in privacy-preserving computations, but, as we show, Annealing sort is much more efficient. We show that there is an input permutation that causes Spin-the-bottle sort to require $\\Omega(n^2\\log n)$ expected time in order to succeed, and that in $O(n^2\\log n)$ time this algorithm succeeds with high probability for any input. We also show there is an implementation of Annealing sort that runs in $O(n\\log n)$ time and succeeds with very high probability.

  14. Spin-the-bottle Sort and Annealing Sort: Oblivious Sorting via Round-robin Random Comparisons.

    Science.gov (United States)

    Goodrich, Michael T

    2014-03-01

    We study sorting algorithms based on randomized round-robin comparisons. Specifically, we study Spin-the-bottle sort, where comparisons are unrestricted, and Annealing sort, where comparisons are restricted to a distance bounded by a temperature parameter. Both algorithms are simple, randomized, data-oblivious sorting algorithms, which are useful in privacy-preserving computations, but, as we show, Annealing sort is much more efficient. We show that there is an input permutation that causes Spin-the-bottle sort to require Ω(n(2) log n) expected time in order to succeed, and that in O(n(2) log n) time this algorithm succeeds with high probability for any input. We also show there is a specification of Annealing sort that runs in O(n log n) time and succeeds with very high probability.

  15. Deductive sort and climbing sort: new methods for non-dominated sorting.

    Science.gov (United States)

    McClymont, Kent; Keedwell, Ed

    2012-01-01

    In recent years an increasing number of real-world many-dimensional optimisation problems have been identified across the spectrum of research fields. Many popular evolutionary algorithms use non-dominance as a measure for selecting solutions for future generations. The process of sorting populations into non-dominated fronts is usually the controlling order of computational complexity and can be expensive for large populations or for a high number of objectives. This paper presents two novel methods for non-dominated sorting: deductive sort and climbing sort. The two new methods are compared to the fast non-dominated sort of NSGA-II and the non-dominated rank sort of the omni-optimizer. The results demonstrate the improved efficiencies of the deductive sort and the reductions in comparisons that can be made when applying inferred dominance relationships defined in this paper.

  16. Selective sorting of waste

    CERN Multimedia

    2007-01-01

    Not much effort needed, just willpower In order to keep the cost of disposing of waste materials as low as possible, CERN provides two types of recipient at the entrance to each building: a green plastic one for paper/cardboard and a metal one for general refuse. For some time now we have noticed, to our great regret, a growing negligence as far as selective sorting is concerned, with, for example, the green recipients being filled with a mixture of cardboard boxes full of polystyrene or protective wrappers, plastic bottles, empty yogurts pots, etc. …We have been able to ascertain, after careful checking, that this haphazard mixing of waste cannot be attributed to the cleaning staff but rather to members of the personnel who unscrupulously throw away their rubbish in a completely random manner. Non-sorted waste entails heavy costs for CERN. For information, once a non-compliant item is found in a green recipient, the entire contents are sent off for incineration rather than recycling… We are all concerned...

  17. Exosome and exosomal microRNA: trafficking, sorting, and function.

    Science.gov (United States)

    Zhang, Jian; Li, Sha; Li, Lu; Li, Meng; Guo, Chongye; Yao, Jun; Mi, Shuangli

    2015-02-01

    Exosomes are 40-100 nm nano-sized vesicles that are released from many cell types into the extracellular space. Such vesicles are widely distributed in various body fluids. Recently, mRNAs and microRNAs (miRNAs) have been identified in exosomes, which can be taken up by neighboring or distant cells and subsequently modulate recipient cells. This suggests an active sorting mechanism of exosomal miRNAs, since the miRNA profiles of exosomes may differ from those of the parent cells. Exosomal miRNAs play an important role in disease progression, and can stimulate angiogenesis and facilitate metastasis in cancers. In this review, we will introduce the origin and the trafficking of exosomes between cells, display current research on the sorting mechanism of exosomal miRNAs, and briefly describe how exosomes and their miRNAs function in recipient cells. Finally, we will discuss the potential applications of these miRNA-containing vesicles in clinical settings.

  18. Exosome and Exosomal MicroRNA: Trafficking, Sorting, and Function

    Directory of Open Access Journals (Sweden)

    Jian Zhang

    2015-02-01

    Full Text Available Exosomes are 40–100 nm nano-sized vesicles that are released from many cell types into the extracellular space. Such vesicles are widely distributed in various body fluids. Recently, mRNAs and microRNAs (miRNAs have been identified in exosomes, which can be taken up by neighboring or distant cells and subsequently modulate recipient cells. This suggests an active sorting mechanism of exosomal miRNAs, since the miRNA profiles of exosomes may differ from those of the parent cells. Exosomal miRNAs play an important role in disease progression, and can stimulate angiogenesis and facilitate metastasis in cancers. In this review, we will introduce the origin and the trafficking of exosomes between cells, display current research on the sorting mechanism of exosomal miRNAs, and briefly describe how exosomes and their miRNAs function in recipient cells. Finally, we will discuss the potential applications of these miRNA-containing vesicles in clinical settings.

  19. The potential of a dielectrophoresis activated cell sorter (DACS) as a next generation cell sorter

    Science.gov (United States)

    Lee, Dongkyu; Hwang, Bohyun; Kim, Byungkyu

    2016-12-01

    Originally introduced by H. A. Pohl in 1951, dielectrophoretic (DEP) force has been used as a striking tool for biological particle manipulation (or separation) for the last few decades. In particular, dielectrophoresis activated cell sorters (DACSes) have been developed for applications in various biomedical fields. These applications include cell replacement therapy, drug screening and medical diagnostics. Since a DACS does not require a specific bio-marker, it is able to function as a biological particle sorting tool with numerous configurations for various cells [e.g. red blood cells (RBCs), white blood cells (WBCs), circulating tumor cells, leukemia cells, breast cancer cells, bacterial cells, yeast cells and sperm cells]. This article explores current DACS capabilities worldwide, and it also looks at recent developments intended to overcome particular limitations. First, the basic theories are reviewed. Then, representative DACSes based on DEP trapping, traveling wave DEP systems, DEP field-flow fractionation and DEP barriers are introduced, and the strong and weak points of each DACS are discussed. Finally, for the purposes of commercialization, prerequisites regarding throughput, efficiency and recovery rates are discussed in detail through comparisons with commercial cell sorters (e.g. fluorescent activated and magnetic activated cell sorters).

  20. Mast cell activation disease

    African Journals Online (AJOL)

    EL-HAKIM

    only IgE dependent allergic diseases but also play a ... Mast cells are tissue fixed effector cells of allergic ..... alleviated high intensity symptoms of MCAD.29 ... Osteoporosis, osteolysis, bone pain: biphosphonates (vitamin D plus calcium.

  1. Determination of telomerase activity in stem cells and non-stem cells of breast cancer

    Institute of Scientific and Technical Information of China (English)

    LI Zhi; HE Yanli; ZHANG Jiahua; ZHANG Jinghui; HUANG Tao

    2007-01-01

    Although all normal tissue cells,including stem cells,are genetically homologous,variation in gene expression patterns has already determined the distinct roles for individual cells in the physiological process due to the occurrence of epigenetic modification.This is of special importance for the existenee of tissue stem cells because they are exclusively immortal within the body,capable of selfreplicating and differentiating by which tissues renew and repair itself and the total tissue cell population maintains a steady-state.Impairment of tissue stem cells is usually accompanied by a reduction in cell number,slows down the repair process and causes hypofunction.For instance,chemotherapy usually leads to depression of bone marrow and hair loss.Cellular aging is closely associated with the continuous erosion of the telomere while activation of telomerase repairs and maintains telomeres,thus slowing the aging process and prolonging cell life.In normal adults,telomerase activation mainly presents in tissue stem cells and progenitor cells giving them unlimited growth potential.Despite the extensive demonstration of telomerase activation in malignancy(>80%),scientists found that heterogeneity also exists among the tumor cells and only minorities of cells,designated as cancer stem cells,andergo processes analogous to the self-renewal and differentiation of normal stem ceils while the rest have limited lifespans.In this study,telomerase activity was measured and compared in breast cancer stem cells and non-stem cells that were phenotypically sorted by examining surface marker expression.The results indicated that cancer stem cells show a higher level of enzyme activity than non-stem cells.In addition,associated with the repair of cancer tissue(or relapse)after chemotherapy,telomerase activity in stem cells was markedly increased.

  2. Phenotypic and functional characterization of earthworm coelomocyte subsets: Linking light scatter-based cell typing and imaging of the sorted populations

    DEFF Research Database (Denmark)

    Engelmann, Péter; Hayashi, Yuya; Bodo, Kornélia;

    2016-01-01

    of lectin binding capacity indicated wheat germ agglutinin (WGA) as the strongest reactor to amoebocytes. This is further evidenced by WGA inhibition assays that suggest high abundance of N-acetyl-d-glucosamine in amoebocytes. Post-sort phagocytosis assays confirmed the functional differences between...

  3. Natural Selection Is a Sorting Process: What Does that Mean?

    Science.gov (United States)

    Price, Rebecca M.

    2013-01-01

    To learn why natural selection acts only on existing variation, students categorize processes as either creative or sorting. This activity helps students confront the misconception that adaptations evolve because species need them.

  4. 乳腺癌MCF-7细胞系中侧群细胞分选及其生物学特性%Sorting of side population cells from breast cancer MCF-7 cell line and its biological characteristics

    Institute of Scientific and Technical Information of China (English)

    孙鑫; 李平; 张梅; 陈娇

    2012-01-01

    Objective To separate the side population cells(SP) from breast cancer MCF-7 cell line,and observe its biological characteristics.Methods Flow cytometry and Hcechst 33342 dye efflux assay were used to isolate SP cells and non-SP cells from the MCF-7 cell line of human breast cancer.Tumorigenicity of the two subpopulations was observed by a soft agar cloning method.Results The results of FACS analysis indicated that (6.5 ± 0.4 ) %of the MCF-7 cells were SP cells;The vitro colony formation rate of SP cells was(38.5 ±9.4)%,and higher than that of non-SP cells ( 8.4 ± 2.6 ) % ( t =5.34,P < 0,05 ).Concluslon The SP cells sorted from MCF-7 cell line enriched tunor stem cells,which exhibited high tumorigenicity.It indicated that SP cells should play a principal role in breast cancer.%目的 分离乳腺癌MCF-7细胞系中的侧群细胞(SP)并观察其生物学特性.方法 利用流式细胞荧光分选法将乳腺癌MCF-7细胞系分成SP和非SP细胞两个亚群.对两个亚群细胞采用软琼脂克隆形成实验观察其增殖能力.结果 MCF-7细胞株中分选出SP细胞占(6.5±0.4)%;SP细胞的体外克隆形成率为(38.5±9.4)%,高于非SP细胞的(8.4±2,6)%(t=5.34,P<0.05).结论 乳腺癌MCF-7细胞中的SP细胞富集了乳腺癌于细胞,其增殖能力强于非SP细胞,表明SP表型的肿瘤细胞在乳腺癌的生长中具有重要的地位.

  5. A Micro Fluorescent Activated Cell Sorter for Astrobiology Applications

    Science.gov (United States)

    Platt, Donald W.; Hoover, Richard B.

    2009-01-01

    A micro-scale Fluorescent Activated Cell Sorter (microFACS) for astrobiology applications is under development. This device is designed to have a footprint of 7 cm x 7 cm x 4 cm and allow live-dead counts and sorting of cells that have fluorescent characteristics from staining. The FACS system takes advantage of microfluidics to create a cell sorter that can fit in the palm of the hand. A micron-scale channel allows cells to pass by a blue diode which causes emission of marker-expressed cells which are detected by a filtered photodetector. A small microcontroller then counts cells and operates high speed valves to select which chamber the cell is collected in (a collection chamber or a waste chamber). Cells with the expressed characteristic will be collected in the collection chamber. This system has been built and is currently being tested. We are also designing a system with integrated MEMS-based pumps and valves for a small and compact unit to fly on small satellite-based biology experiments.

  6. 免疫磁珠法在分离纯化外周血CD4+和CD8+T淋巴细胞亚群中的应用%APPLICATION OF MAGNETIC ACTIVARED CELL SORTING FOR SEPARATION AND PURIFICATION OF CD4+ T CELL AND CD8+T CELL SUBPOPULATIONS OF PERIPHERAL BLOOD

    Institute of Scientific and Technical Information of China (English)

    莫雪安; 周礼圆; 秦超; 张德敏; 赵伟金

    2012-01-01

    目的:探讨免疫磁珠法(MiniMACS)在分离纯化外周血CD4+和CD8+T淋巴细胞亚群中的应用.方法:应用密度梯度离心法分离外周血单个核细胞(Peripheral blood mononucleate cells,PBMC),采用MiniMACS分别分离和纯化39例标本外周血PBMC中的CD4+T淋巴细胞和CD8+T淋巴细胞,并经流式细胞仪分析细胞纯度和台盼蓝染色的方法对细胞活力进行评估.结果:MiniMACS分离外周血CD4+T淋巴细胞前、后细胞纯度分别为(37.38±5.74)%、(97.75±1.03)%(P<0.001),CD8+T淋巴细胞分离纯化前后细胞纯度分别为(20.11±6.83)%、(96.85±1.86)%(P<0.001);外周血分离前PBMC细胞活力为(97.66±2.73)%,纯化为CD4+T淋巴细胞和CD8+T淋巴细胞后细胞活力分别为(97.44±3.08)%、(98.05±2.92)%(P>0.05).结论:MiniMACS可以高度富集CD4+T淋巴细胞和CD8+T淋巴细胞且不改变细胞的活力.%To evaluate the separation and purification of T lymphocyte subsets by immunomag-netic beads. Methods: Peripheral blood mononucleate cells (PBMC) were isolated using lymphocytes separation medium and desity gradient centrifugal action. CD4+T and CD8+T lymphocytes were isolated and purified from PBMC by magnetic activated cell sorting (MiniMACS). The purity and activity of CD4+T and CD8+T cells were measured using flow cytometry, trypan-blue dye exclusion test, etc. Results: The results showed that the percentage of CD4+T cells before and after the purification was (37. 38 + 5. 74)% and (97. 75 + 1. 03)% ( P <0. 001) ; the percentage of CD8+T cells before and after the purification was (20. 11 + 6. 83) % and (96. 85 + 1. 86) % ( P <0. 001) , resprectively; and the cell ability was not affected by purification procedure ( P <0. 05). Conclusion: MiniMACS can sort out effectively CD4 + T and CD8+ T cell subpopulations of peripheral blood and does not affect the cell vitality.

  7. Interplay of Endosomal pH and Ligand Occupancy in Integrin α5β1 Ubiquitination, Endocytic Sorting, and Cell Migration

    Directory of Open Access Journals (Sweden)

    Dmitri Kharitidi

    2015-10-01

    Full Text Available Membrane trafficking of integrins plays a pivotal role in cell proliferation and migration. How endocytosed integrins are targeted either for recycling or lysosomal delivery is not fully understood. Here, we show that fibronectin (FN binding to α5β1 integrin triggers ubiquitination and internalization of the receptor complex. Acidification facilitates FN dissociation from integrin α5β1 in vitro and in early endosomes, promoting receptor complex deubiquitination by the USP9x and recycling to the cell surface. Depending on residual ligand occupancy of receptors, some α5β1 integrins remain ubiquitinated and are captured by ESCRT-0/I, containing histidine domain-containing protein tyrosine phosphatase (HD-PTP and ubiquitin-associated protein 1 (UBAP1, and are directed for lysosomal proteolysis, limiting receptor downstream signaling and cell migration. Thus, HD-PTP or UBAP1 depletion confers a pro-invasive phenotype. Thus, pH-dependent FN-integrin dissociation and deubiquitination of the activated integrin α5β1 are required for receptor resensitization and cell migration, representing potential targets to modulate tumor invasiveness.

  8. Microfluidic droplet sorting using integrated bilayer micro-valves

    Science.gov (United States)

    Chen, Yuncong; Tian, Yang; Xu, Zhen; Wang, Xinran; Yu, Sicong; Dong, Liang

    2016-10-01

    This paper reports on a microfluidic device capable of sorting microfluidic droplets utilizing conventional bilayer pneumatic micro-valves as sorting controllers. The device consists of two micro-valves placed symmetrically on two sides of a sorting area, each on top of a branching channel at an inclined angle with respect to the main channel. Changes in transmitted light intensity, induced by varying light absorbance by each droplet, are used to divert the droplet from the sorting area into one of the three outlet channels. When no valve is activated, the droplet flows into the outlet channel in the direction of the main channel. When one of the valves is triggered, the flexible membrane of valve will first be deflected. Once the droplet leaves the detection point, the deflected membrane will immediately return to its default flattened position, thereby exerting a drawing pressure on the droplet and deviating it from its original streamline to the outlet on the same side as the valve. This sorting method will be particularly suitable for numerous large-scale integrated microfluidic systems, where pneumatic micro-valves are already used. Only few structural modifications are needed to achieve droplet sorting capabilities in these systems. Due to the mechanical nature of diverting energy applied to droplets, the proposed sorting method may induce only minimal interference to biological species or microorganisms encapsulated inside the droplets that may accompany electrical, optical and magnetic-based techniques.

  9. 免疫磁珠法分离膀胱癌CD133+细胞及生物学行为研究%Isolation of CD133 positive bladder cancer cells with magnetic cell sorting system and research on the biological characteristics

    Institute of Scientific and Technical Information of China (English)

    李法平; 陈帅奇; 王艳波; 郭辉; 刘二鹏; 侯宇川

    2014-01-01

    目的 探讨免疫磁珠法分离膀胱癌5637细胞株中CD133+细胞的方法,观察CD133阳性、阴性细胞间生物学行为的差异.方法 采用免疫磁珠法分选出膀胱癌5637细胞株中CD133+细胞,流式细胞仪检测分选纯度,通过噻唑蓝(MTT)实验、平板克隆形成实验、细胞划痕实验、分化能力检测研究其生物学行为.结果 流式细胞仪检测CD133+细胞在膀胱癌5637细胞中比例为1.45%,经免疫磁珠法分选所得CD133+细胞比例为93.45%;CD133+细胞的增殖、迁移能力明显强于CD133-、未分选的肿瘤细胞(P<0.05);平板克隆形成实验结果示CD133+组[(89.333±4.530)%]细胞克隆形成率明显高于CD133-组[(22.667±4.041)%,P<0.05].流式细胞仪检测CD133+细胞培养2d后其纯度降至48.19%,4d后其纯度与分选前无明显差异.结论 免疫磁珠分选技术可高效快捷获得CD133+细胞,CD133+细胞具有自我更新、迁徙、生成其他表型肿瘤细胞等干细胞样特性.%Objective To isolate CD133 positive cell from bladder cancer cell line 5637 with magnetic cell sorting system (MACS) and to study the different biological characteristics of CD133+ and CD133 cells.Methods Flow cytometry was used to determine the proportion of CD133 + cells sorted by magnetic-activated cell sorting in the bladder cancer cell line 5637.The biological characteristics of CD133 + and CD133-cells was studied by methyl thiazol tetrazolium (MTT) assay,Flat colony formation,Wound healing assay and differentiation ability.Results The proportion of CD133 + cells in the bladder cancer cell line 5637 was 1.45%,which was determined by flow cytometry.CD133 + cells purified by MACS were in a considerable purity of 93.42%.The proliferation and migration capacity of CD133 + cells display stronger than that of CD133-cells and non-separated tumor cells (P <0.05).The formation rate of colony sphere in CD133+ group [(89.333 ± 4.530)%] was higher than that in CD133-group

  10. Sorting and selection in posets

    DEFF Research Database (Denmark)

    Daskalakis, Constantinos; Karp, Richard M.; Mossel, Elchanan

    2011-01-01

    Classical problems of sorting and searching assume an underlying linear ordering of the objects being compared. In this paper, we study these problems in the context of partially ordered sets, in which some pairs of objects are incomparable. This generalization is interesting from a combinatorial...

  11. A Radar Active Jamming Sorting Based on Feature Weighted and Support Vector Machine%基于特征加权与SVM的雷达有源干扰分类技术

    Institute of Scientific and Technical Information of China (English)

    唐翥; 张兵; 李广强; 沈浩浩

    2014-01-01

    In order to improve the the active jamming sorting accuracy effectively,a sorting method based on feature weighted and support vector machine is proposed. The feature weighting concept according to the different importance degree of each signal feature parameters to signal classification in the process is introduced. Using the gray relational analysis to obtain the weight of each feature,and some weak characteristics for the huge impact on the classification results are avoided. Finally,using support vector machine classifier,the active radar interference signal is classified and identified. Simulation experiments show that this method can improve the recognition rate of the radar active interference signal type effectively.%为了有效提高雷达有源干扰分类正确率,提出一种基于特征加权与支持向量机的分类方法。针对分类过程中各信号特征参数对信号分类的重要度不同,引入特征加权的概念。利用灰色关联分析方法求取各特征权重,避免一些弱特征对分类结果产生较大影响。最后利用支持向量机分类器,对雷达有源干扰信号进行了分类识别。通过仿真实验证明,该方法可以有效提高雷达有源干扰信号类型的识别率,具有很好的通用性。

  12. Molecular characterization of flow-sorted mammalian centromeres

    Energy Technology Data Exchange (ETDEWEB)

    Hamkalo, B.A.; Henschen, A.; Parseghian, M.H. [Univ. of Calfornia, Irvine, CA (United States). Dept. of Molecular Biology and Biochemistry] [and others

    1998-12-31

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The project involved experiments directed towards developing a molecular characterization of the centromere region of mammalian chromosomes. Attempts to purify this essential chromosomal locus by conventional methods have thus far been unsuccessful. However, preliminary data obtained in collaboration with the National Flow Cytometry Resource (NFCR) showed that it is possible to purify a chromosome fragment that is present in certain cultured mouse cell lines and has all the properties expected of an intact centromere region. To begin sorting this minichromosome for the identification of proteins preferentially associated with centromere regions, standard buffers utilized in chromosome sorting were evaluated for potential effects on maintenance of chromosomal proteins during sorting. The data indicate that the presence of several buffer constituents results in the extraction of all but a few chromosomal proteins. The subsequent use of a magnesium sulfate buffer resulted in the sorting of mouse chromosomes that do not suffer a significant loss of proteins. Several DNA stains were also evaluated for causing protein dissociation, but no significant losses were observed. Although flow-sorted chromosomes have been used extensively for DNA analysis and cloning, this is a pioneering effort by the NFCR, and its collaborators, to exploit chromosome sorting capabilities for the analysis of chromosomal proteins.

  13. Molecular characterization of flow-sorted mammalian centromeres

    Energy Technology Data Exchange (ETDEWEB)

    Hamkalo, B.A.; Henschen, A.; Parseghian, M.H. [Univ. of Calfornia, Irvine, CA (United States). Dept. of Molecular Biology and Biochemistry] [and others

    1998-12-31

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The project involved experiments directed towards developing a molecular characterization of the centromere region of mammalian chromosomes. Attempts to purify this essential chromosomal locus by conventional methods have thus far been unsuccessful. However, preliminary data obtained in collaboration with the National Flow Cytometry Resource (NFCR) showed that it is possible to purify a chromosome fragment that is present in certain cultured mouse cell lines and has all the properties expected of an intact centromere region. To begin sorting this minichromosome for the identification of proteins preferentially associated with centromere regions, standard buffers utilized in chromosome sorting were evaluated for potential effects on maintenance of chromosomal proteins during sorting. The data indicate that the presence of several buffer constituents results in the extraction of all but a few chromosomal proteins. The subsequent use of a magnesium sulfate buffer resulted in the sorting of mouse chromosomes that do not suffer a significant loss of proteins. Several DNA stains were also evaluated for causing protein dissociation, but no significant losses were observed. Although flow-sorted chromosomes have been used extensively for DNA analysis and cloning, this is a pioneering effort by the NFCR, and its collaborators, to exploit chromosome sorting capabilities for the analysis of chromosomal proteins.

  14. The cell biology of T-dependent B cell activation

    DEFF Research Database (Denmark)

    Owens, T; Zeine, R

    1989-01-01

    The requirement that CD4+ helper T cells recognize antigen in association with class II Major Histocompatibility Complex (MHC) encoded molecules constrains T cells to activation through intercellular interaction. The cell biology of the interactions between CD4+ T cells and antigen-presenting cells...... activation through coculture with T cells activated by anti-T-cell receptor or anti-CD3 antibodies suggest that cellular interaction with T cells, independent of antigen presentation or lymphokine secretion, induces or triggers B cells to become responsive to T-derived lymphokines, and that this may...

  15. Boar sperm changes after sorting and encapsulation in barium alginate membranes.

    Science.gov (United States)

    Spinaci, M; Bucci, D; Chlapanidas, T; Vallorani, C; Perteghella, S; Communod, R; Vigo, D; Tamanini, C; Galeati, G; Faustini, M; Torre, M L

    2013-09-15

    A routine use of boar-sexed semen is limited by the long sorting time necessary to obtain an adequate number of sexed spermatozoa for artificial insemination and by the high susceptibility of spermatozoa of this species to damages induced by sorting procedure and subsequent cryopreservation. The aim of this work was to study the impact of encapsulation in barium alginate membrane on sorted boar spermatozoa by evaluating membrane integrity, chlortetracycline staining patterns, protein tyrosine phosphorylation, and Hsp70 immunolocalization during storage over 72 hours in liquid or encapsulated form. The encapsulation procedure significantly (P < 0.05) decreased the overall membrane integrity of control unsorted semen (81.8 vs. 57.4, CTR vs. CPS), but did not negatively affect the overall viability and the chlortetracycline staining patterns of sorted encapsulated cells. Moreover, encapsulation significantly decreased (P < 0.05) the overall phosphotyrosin A pattern cell percentage in unsorted (98.4 vs. 92.6, CTR vs. CPS) but not in sorted semen (64.0 vs. 74.2; SORT CTR vs. SORT CPS). As for Hsp70, the overall percentage of cells displaying the different patterns was significantly influenced (P < 0.05) by treatment but not by storage time. The sorting procedure seems to induce the major changes, whereas encapsulation tends to exert a protective effect on sorted semen by increasing the percentage of spermatozoa displaying the T pattern (2.8 vs. 24.3; SORT CTR vs. SORT CPS). In conclusion, our data confirm that the damaging impact of the encapsulation in barium alginate capsules seems to be limited when compared with that of the sorting procedure and, moreover, the association of the two procedures does not result in an algebraic sum of the negative effects. These results suggest the possibility of a future utilization of the encapsulation technology in order to store sorted spermatozoa and permit their controlled release in the female genital tract.

  16. Swarm-Based Spatial Sorting

    CERN Document Server

    Amos, Martyn

    2008-01-01

    Purpose: To present an algorithm for spatially sorting objects into an annular structure. Design/Methodology/Approach: A swarm-based model that requires only stochastic agent behaviour coupled with a pheromone-inspired "attraction-repulsion" mechanism. Findings: The algorithm consistently generates high-quality annular structures, and is particularly powerful in situations where the initial configuration of objects is similar to those observed in nature. Research limitations/implications: Experimental evidence supports previous theoretical arguments about the nature and mechanism of spatial sorting by insects. Practical implications: The algorithm may find applications in distributed robotics. Originality/value: The model offers a powerful minimal algorithmic framework, and also sheds further light on the nature of attraction-repulsion algorithms and underlying natural processes.

  17. Sorting fluorescent nanocrystals with DNA

    Energy Technology Data Exchange (ETDEWEB)

    Gerion, Daniele; Parak, Wolfgang J.; Williams, Shara C.; Zanchet, Daniela; Micheel, Christine M.; Alivisatos, A. Paul

    2001-12-10

    Semiconductor nanocrystals with narrow and tunable fluorescence are covalently linked to oligonucleotides. These biocompounds retain the properties of both nanocrystals and DNA. Therefore, different sequences of DNA can be coded with nanocrystals and still preserve their ability to hybridize to their complements. We report the case where four different sequences of DNA are linked to four nanocrystal samples having different colors of emission in the range of 530-640 nm. When the DNA-nanocrystal conjugates are mixed together, it is possible to sort each type of nanoparticle using hybridization on a defined micrometer -size surface containing the complementary oligonucleotide. Detection of sorting requires only a single excitation source and an epifluorescence microscope. The possibility of directing fluorescent nanocrystals towards specific biological targets and detecting them, combined with their superior photo-stability compared to organic dyes, opens the way to improved biolabeling experiments, such as gene mapping on a nanometer scale or multicolor microarray analysis.

  18. Sorting Techniques for Plastics Recycling

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    This paper presents the basic principles of three different types of separating methods and a general guideline for choosing the most effective method for sorting plastic mixtures. It also presents the results of the tests carried out for separation of PVC, ABS and PET from different kinds of plastic mixtures in order to improve the grade of the raw input used in mechanical or feedstock recycling.

  19. Sorting of Sperm by Morphology

    Science.gov (United States)

    Koh, James; Marcos, Marcos

    2016-11-01

    Many studies have proven that the percentage of morphologically normal sperm is a significant factor in determining the success of assisted reproduction. The velocity of sperm in a microchannel with shear flow subjected to an external field will be explored theoretically. The difference in response between morphologically normal and abnormal sperm will be computed from a statistical approach, to study the feasibility and effectiveness of sorting by an external field to remove abnormal sperm. The full name of this author is Marcos.

  20. Self-sorting molecular clips.

    Science.gov (United States)

    Ghosh, Soumyadip; Wu, Anxin; Fettinger, James C; Zavalij, Peter Y; Isaacs, Lyle

    2008-08-01

    We report the synthesis and characterization of 12 C-shaped methylene-bridged glycoluril dimers (1-12) bearing H-bonding groups on their aromatic rings. Compounds 1, 2, (+/-)-4a, (+/-)-5, (+/-)-7, and 8 form tightly associated homodimers in CDCl3, due to the combined driving force of pi-pi and H-bonding interactions. Compounds 2, (+/-)-5, and 8, having disparate spatial distribution of their H-bonding groups, display the ability to efficiently distinguish between self and nonself even within three-component mixtures in CDCl3. When the spatial distributions of the H-bonding groups of the molecular clips are similar (e.g., 1 and 2), a mixture of homodimers and heterodimers is formed. The effect of various structural modifications (e.g., chirality, side chain steric bulk, number and pattern of H-bonds) on the strength of self-assembly and the fidelity of self-sorting are presented. On the basis of these results we prepared self-sorting systems comprising three (e.g., 1, (+/-)-5, and (+/-)-7) and even four ( 2, (+/-)-5, 9, and 10) components. The potential of molecular clips 1-12 as robust, functionalizable, self-sorting modules to control the noncovalent interaction network in systems chemistry studies is described.

  1. Schwann cell myelination requires integration of laminin activities.

    Science.gov (United States)

    McKee, Karen K; Yang, Dong-Hua; Patel, Rajesh; Chen, Zu-Lin; Strickland, Sidney; Takagi, Junichi; Sekiguchi, Kiyotoshi; Yurchenco, Peter D

    2012-10-01

    Laminins promote early stages of peripheral nerve myelination by assembling basement membranes (BMs) on Schwann cell surfaces, leading to activation of β1 integrins and other receptors. The BM composition, structural bonds and ligands needed to mediate this process, however, are not well understood. Mice hypomorphic for laminin γ1-subunit expression that assembled endoneurial BMs with reduced component density exhibited an axonal sorting defect with amyelination but normal Schwann cell proliferation, the latter unlike the null. To identify the basis for this, and to dissect participating laminin interactions, LAMC1 gene-inactivated dorsal root ganglia were treated with recombinant laminin-211 and -111 lacking different architecture-forming and receptor-binding activities, to induce myelination. Myelin-wrapping of axons by Schwann cells was found to require higher laminin concentrations than either proliferation or axonal ensheathment. Laminins that were unable to polymerize through deletions that removed critical N-terminal (LN) domains, or that lacked cell-adhesive globular (LG) domains, caused reduced BMs and almost no myelination. Laminins engineered to bind weakly to α6β1 and/or α7β1 integrins through their LG domains, even though they could effectively assemble BMs, decreased myelination. Proliferation depended upon both integrin binding to LG domains and polymerization. Collectively these findings reveal that laminins integrate scaffold-forming and cell-adhesion activities to assemble an endoneurial BM, with myelination and proliferation requiring additional α6β1/α7β1-laminin LG domain interactions, and that a high BM ligand/structural density is needed for efficient myelination.

  2. Effect evaluation on sorting method of CD4+T lymphocytes%CD4+T淋巴细胞分选方法的效果评价

    Institute of Scientific and Technical Information of China (English)

    张艳; 王记红

    2014-01-01

    Objective To establish an accurate sorting method with less interference to the cell activity for isolating CD4+T lymphocytes.Methods Human peripheral blood cells were sorted by density gradient centrifugation (DGC) and magnetic activated cell sorting (MACS) in turn,and then the cell purity,morphological observation and survival rate of CD4+T lymphocytes were adopted to conduct the evaluation on the sorting method.Results The purity of CD4+T lymphocytes was (38.8 ± 2.7)%,while the purity of CD4+T lymphocytes was (96.2 ± 0.7)% after magnetic activated cells being sorted,the difference between them was statistically significant (P < 0.01).The activity was the highest within 24 hours with intact cell shape and function.Conclusion The Percoll DGC combined with the MACS can collect highly pure CD4+T lymphocytes with less influence on the cell activity and shape,the sorted cells can continue to be used for related researches.%目的 建立一种准确且对细胞活性影响小的分离人外周血CD4+T细胞的分选方法.方法 对人外周血依次采用密度梯度离心(density gradient centrifugation,DGC)和免疫磁珠分选(magnetic-activated cell sorting,MACS)进行CD4+T细胞分选.分选后的细胞用流式细胞仪(flow cytometry,FCM)、倒置显微镜及细胞周期分析进行分选纯度和生长状态检测.结果 Percoll密度梯度离心法收集的单个核细胞中CD4+T细胞纯度为(38.8±2.7)%,免疫磁珠分选后CD4+T细胞纯度为(96.2±0.7)%,两者比较差异有统计学意义(P<0.01),且分选后细胞形态及功能完好,24 h内活性最高.结论 密度梯度离心和免疫磁珠分选相结合可以有效提高CD4+T细胞的分选纯度及保持其活性,可继续用于相关功能研究.

  3. How does the Shift-insertion sort behave when the sorting elements follow a Normal distribution?

    CERN Document Server

    Pal, Mita; Mahanti, N C

    2012-01-01

    The present paper examines the behavior of Shift-insertion sort (insertion sort with shifting) for normal distribution inputs and is in continuation of our earlier work on this new algorithm for discrete distribution inputs, namely, negative binomial. Shift insertion sort is found more sensitive for main effects but not for all interaction effects compared to conventional insertion sort.

  4. Curcumin reduces expression of Bcl-2, leading to apoptosis in daunorubicin-insensitive CD34+ acute myeloid leukemia cell lines and primary sorted CD34+ acute myeloid leukemia cells

    Science.gov (United States)

    2011-01-01

    Background Acute myeloid leukemia (AML) is an immunophenotypically heterogenous malignant disease, in which CD34 positivity is associated with poor prognosis. CD34+ AML cells are 10-15-fold more resistant to daunorubicin (DNR) than CD34- AML cells. Curcumin is a major component of turmeric that has shown cytotoxic activity in multiple cancers; however, its anti-cancer activity has not been well studied in DNR-insensitive CD34+ AML cells. The aim of this study was to therefore to explore curcumin-induced cytotoxicity in DNR-insensitive CD34+ AML cell lines (KG1a, Kasumi-1), DNR-sensitive U937 AML cells, and primary CD34+ AML bone-marrow-derived cells. Methods Primary human CD34+ cells were isolated from peripheral blood mononuclear cells or bone marrow mononuclear cells using a CD34 MicroBead kit. The growth inhibitory effects of curcumin were evaluated by MTT and colony-formation assays. Cell cycle distribution was examined by propidium iodide (PI) assay. Apoptosis was analyzed by Wright-Giemsa, Hoechst 33342 and Annexin-V/PI staining assays. The change in mitochondrial membrane potential (MMP) was examined by JC-1 staining and flow cytometry. Expression of apoptosis-related proteins was determined by reverse transcription-polymerase chain reaction and Western blotting. Short interfering RNA (siRNA) against Bcl-2 was used in CD34+ KG1a and Kasumi-1 cells incubated with/without DNR. Results Curcumin inhibited proliferation and induced apoptosis and G1/S arrest in both DNR-insensitive KG1a, Kasumi-1 and DNR-sensitive U937 cells. Curcumin-induced apoptosis was associated with reduced expression of both Bcl-2 mRNA and protein, subsequent loss of MMP, and activation of caspase-3 followed by PARP degradation. Curcumin synergistically enhanced the cytotoxic effect of DNR in DNR-insensitive KG1a and Kasumi-1 cells, consistent with decreased Bcl-2 expression. Accordingly, siRNA against Bcl-2 increased the susceptibility of KG1a and Kasumi-1 cells to DNR-induced apoptosis

  5. Curcumin reduces expression of Bcl-2, leading to apoptosis in daunorubicin-insensitive CD34+ acute myeloid leukemia cell lines and primary sorted CD34+ acute myeloid leukemia cells

    Directory of Open Access Journals (Sweden)

    Huang Sheng-Shan

    2011-05-01

    Full Text Available Abstract Background Acute myeloid leukemia (AML is an immunophenotypically heterogenous malignant disease, in which CD34 positivity is associated with poor prognosis. CD34+ AML cells are 10-15-fold more resistant to daunorubicin (DNR than CD34- AML cells. Curcumin is a major component of turmeric that has shown cytotoxic activity in multiple cancers; however, its anti-cancer activity has not been well studied in DNR-insensitive CD34+ AML cells. The aim of this study was to therefore to explore curcumin-induced cytotoxicity in DNR-insensitive CD34+ AML cell lines (KG1a, Kasumi-1, DNR-sensitive U937 AML cells, and primary CD34+ AML bone-marrow-derived cells. Methods Primary human CD34+ cells were isolated from peripheral blood mononuclear cells or bone marrow mononuclear cells using a CD34 MicroBead kit. The growth inhibitory effects of curcumin were evaluated by MTT and colony-formation assays. Cell cycle distribution was examined by propidium iodide (PI assay. Apoptosis was analyzed by Wright-Giemsa, Hoechst 33342 and Annexin-V/PI staining assays. The change in mitochondrial membrane potential (MMP was examined by JC-1 staining and flow cytometry. Expression of apoptosis-related proteins was determined by reverse transcription-polymerase chain reaction and Western blotting. Short interfering RNA (siRNA against Bcl-2 was used in CD34+ KG1a and Kasumi-1 cells incubated with/without DNR. Results Curcumin inhibited proliferation and induced apoptosis and G1/S arrest in both DNR-insensitive KG1a, Kasumi-1 and DNR-sensitive U937 cells. Curcumin-induced apoptosis was associated with reduced expression of both Bcl-2 mRNA and protein, subsequent loss of MMP, and activation of caspase-3 followed by PARP degradation. Curcumin synergistically enhanced the cytotoxic effect of DNR in DNR-insensitive KG1a and Kasumi-1 cells, consistent with decreased Bcl-2 expression. Accordingly, siRNA against Bcl-2 increased the susceptibility of KG1a and Kasumi-1 cells to

  6. Mechanisms of cell propulsion by active stresses

    Energy Technology Data Exchange (ETDEWEB)

    Carlsson, A E, E-mail: aec@wustl.edu [Department of Physics, Washington University, Campus Box 1105, One Brookings Drive, St. Louis, MO 63130 (United States)

    2011-07-15

    The mechanisms by which cytoskeletal flows and cell-substrate interactions interact to generate cell motion are explored by using a simplified model of the cytoskeleton as a viscous gel containing active stresses. This model yields explicit general results relating cell speed and traction forces to the distributions of active stress and cell-substrate friction. It is found that (i) the cell velocity is given by a function that quantifies the asymmetry of the active-stress distribution, (ii) gradients in cell-substrate friction can induce motion even when the active stresses are symmetrically distributed, (iii) the traction-force dipole is enhanced by protrusive stresses near the cell edges or contractile stresses near the center of the cell and (iv) the cell velocity depends biphasically on the cell-substrate adhesion strength if active stress is enhanced by adhesion. Specific experimental tests of the calculated dependences are proposed.

  7. The cell biology of T-dependent B cell activation

    DEFF Research Database (Denmark)

    Owens, T; Zeine, R

    1989-01-01

    The requirement that CD4+ helper T cells recognize antigen in association with class II Major Histocompatibility Complex (MHC) encoded molecules constrains T cells to activation through intercellular interaction. The cell biology of the interactions between CD4+ T cells and antigen-presenting cells...... includes multipoint intermolecular interactions that probably involve aggregation of both polymorphic and monomorphic T cell surface molecules. Such aggregations have been shown in vitro to markedly enhance and, in some cases, induce T cell activation. The production of T-derived lymphokines that have been...... implicated in B cell activation is dependent on the T cell receptor for antigen and its associated CD3 signalling complex. T-dependent help for B cell activation is therefore similarly MHC-restricted and involves T-B intercellular interaction. Recent reports that describe antigen-independent B cell...

  8. Evidence for local dendritic cell activation in pulmonary sarcoidosis

    Directory of Open Access Journals (Sweden)

    Berge Bregje

    2012-04-01

    Full Text Available Abstract Background Sarcoidosis is a granulomatous disease characterized by a seemingly exaggerated immune response against a difficult to discern antigen. Dendritic cells (DCs are pivotal antigen presenting cells thought to play an important role in the pathogenesis. Paradoxically, decreased DC immune reactivity was reported in blood samples from pulmonary sarcoidosis patients. However, functional data on lung DCs in sarcoidosis are lacking. We hypothesized that at the site of disease DCs are mature, immunocompetent and involved in granuloma formation. Methods We analyzed myeloid DCs (mDCs and plasmacytoid DCs (pDCs in broncho-alveolar lavage (BAL and blood from newly diagnosed, untreated pulmonary sarcoidosis patients and healthy controls using 9-color flowcytometry. DCs, isolated from BAL using flowcytometric sorting (mDCs or cultured from monocytes (mo-DCs, were functionally assessed in a mixed leukocyte reaction with naïve allogeneic CD4+ T cells. Using Immunohistochemistry, location and activation status of CD11c+DCs was assessed in mucosal airway biopsies. Results mDCs in BAL, but not in blood, from sarcoidosis patients were increased in number when compared with mDCs from healthy controls. mDCs purified from BAL of sarcoidosis patients induced T cell proliferation and differentiation and did not show diminished immune reactivity. Mo-DCs from patients induced increased TNFα release in co-cultures with naïve allogeneic CD4+ T cells. Finally, immunohistochemical analyses revealed increased numbers of mature CD86+ DCs in granuloma-containing airway mucosal biopsies from sarcoidosis patients. Conclusion Taken together, these finding implicate increased local DC activation in granuloma formation or maintenance in pulmonary sarcoidosis.

  9. A negative dielectrophoresis and gravity-driven flow-based high-throughput and high-efficiency cell-sorting system.

    Science.gov (United States)

    Lee, Dongkyu; Kim, Dowon; Kim, Youngwoong; Park, Ki-Hyun; Oh, Eun-Jee; Kim, Yonggoo; Kim, Byungkyu

    2014-02-01

    We present a negative dielectrophoresis (n-DEP)-based cell separation system for high-throughput and high-efficiency cell separation. To achieve a high throughput, the proposed system comprises macro-sized channel and cantilever-type electrode (CE) arrays (L × W × H = 150 µm × 500 µm × 50 µm) to generate n-DEP force. For high efficiency, double separation modules, which have macro-sized channels and CE arrays in each separation module, are employed. In addition, flow regulators to precisely control the hydrodynamic force are allocated for each outlet. Because the hydrodynamic force and the n-DEP force acting on the target cell are the main determinants of the separation efficiency, we evaluate the theoretical amount of hydrodynamic force and n-DEP force acting on each target cell. Based on theoretical results, separation conditions are experimentally investigated. Finally, to demonstrate the separation performance, we performed the separation of target cells (live K562) from nontarget cells (dead K562) under conditions of low voltage (7Vp-p with 100 kHz) and a flow rate of 15 µL•min⁻¹, 6 µL•min⁻¹, and 8 µL•min⁻¹ in outlets 1, 2, and 3, respectively. The system can separate target cells with 95% separation efficiency in the case of the ratio of 5:1 (live K562:dead K562).

  10. Expanded Hematopoietic Progenitor Cells Reselected for High Aldehyde Dehydrogenase Activity Demonstrate Islet Regenerative Functions.

    Science.gov (United States)

    Seneviratne, Ayesh K; Bell, Gillian I; Sherman, Stephen E; Cooper, Tyler T; Putman, David M; Hess, David A

    2016-04-01

    Human umbilical cord blood (UCB) hematopoietic progenitor cells (HPC) purified for high aldehyde dehydrogenase activity (ALDH(hi) ) stimulate islet regeneration after transplantation into mice with streptozotocin-induced β cell deletion. However, ALDH(hi) cells represent a rare progenitor subset and widespread use of UCB ALDH(hi) cells to stimulate islet regeneration will require progenitor cell expansion without loss of islet regenerative functions. Here we demonstrate that prospectively purified UCB ALDH(hi) cells expand efficiently under serum-free, xeno-free conditions with minimal growth factor supplementation. Consistent with the concept that ALDH-activity is decreased as progenitor cells differentiate, kinetic analyses over 9 days revealed the frequency of ALDH(hi) cells diminished as culture time progressed such that total ALDH(hi) cell number was maximal (increased 3-fold) at day 6. Subsequently, day 6 expanded cells (bulk cells) were sorted after culture to reselect differentiated progeny with low ALDH-activity (ALDH(lo) subset) from less differentiated progeny with high ALDH-activity (ALDH(hi) subset). The ALDH(hi) subset retained primitive cell surface marker coexpression (32.0% ± 7.0% CD34(+) /CD38(-) cells, 37.0% ± 6.9% CD34(+) /CD133(+) cells), and demonstrated increased hematopoietic colony forming cell function compared with the ALDH(lo) subset. Notably, bulk cells or ALDH(lo) cells did not possess the functional capacity to lower hyperglycemia after transplantation into streptozotocin-treated NOD/SCID mice. However, transplantation of the repurified ALDH(hi) subset significantly reduced hyperglycemia, improved glucose tolerance, and increased islet-associated cell proliferation and capillary formation. Thus, expansion and delivery of reselected UCB cells that retain high ALDH-activity after short-term culture represents an improved strategy for the development of cellular therapies to enhance islet regeneration in situ.

  11. Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting: Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4{sup +} T cells compared

    Energy Technology Data Exchange (ETDEWEB)

    Ford, A.L.; Goodsall, A.L.; Sedgwick, J.D. [Centenary Institute of Cancer Medicine and Cell Biology, Sydney (Australia)] [and others

    1995-05-01

    Ramified microglia in the adult central nervous system (CNS) are the principal glial element up-regulating MHC class I and II expression in response to inflammatory events or neuronal damage. A proportion of these cells also express MHC class II constitutively in the normal CNS. The role of microglia as APCs for CD4{sup +} cells extravasating into the CNS remains undefined. In this study, using irradiation bone marrow chimeras in CD45-congenic rats, the phenotype CD45{sup low}CD11b/c{sup +} is shown to identify microglial cells specifically within the CNS. Highly purified populations of microglia and nonmicroglial but CNS-associated macrophages (CD45{sup high}CD11b/c{sup +}) have been obtained directly from the adult CNS, by using flow cytometric sorting. Morphologically, freshly isolated microglia vs other CNS macrophages are quite distinct. Of the two populations recovered from the normal CNS, it is the minority CD45{sup high}CD11 b/c{sup +} transitional macrophage population, and not microglia, that is the effective APC for experimental autoimmune encephalomyelitis-inducing CD4{sup +} myelin basic protein (MBP)-reactive T cells. CD45{sup high}CD11b/c{sup +} CNS macrophages also stimulate MBP-reactive T cells without addition of MBP to culture suggesting presentation of endogenous Ag. This is the first study in which microglia vs other CNS macrophages have been analyzed for APC ability directly from the CNS, with substantial cross-contamination between the two populations eliminated. The heterogeneity of these populations in terms of APC function is clearly demonstrated. Evidence is still lacking that adult CNS microglia have the capacity to interact with and stimulate CD4{sup +} T cells to proliferate or secrete IL-2. 60 refs., 6 figs., 1 tab.

  12. 磁珠细胞分选CD133+/CD44+前列腺癌干细胞的初步鉴定%Initial identification of CD133+/CD44+ prostate cancer stem cell through magnetic bead cell sorting

    Institute of Scientific and Technical Information of China (English)

    盛夏; 王德林; 李文宾; 罗照

    2013-01-01

    目的:通过磁珠细胞分选(magnetic bead cell sorting,MACS)方法从人前列腺癌细胞系PC-3中分选CD133+/CD44+干细胞,为进一步功能性研究奠定基础.方法:运用流式细胞仪(flow cytometry,FCM)检测MACS分选前后PC-3细胞膜上CD133和CD44表达情况;观察无血清培养成球情况,免疫荧光(immunofluorescenee,IF)表达情况;比较细胞在分选前后的形态学、增殖能力方面的差异;免疫组化(immunohistochemistry,IHC)和Western blot检测诱导分化前后前列腺酸性磷酸酶(prostatic acid phosphatase,PAP)蛋白情况.结果:FCM检测PC-3细胞CD133和CD44的阳性表达分别是(1.33±0.05)%和(0.87±0.06)%,而MACS分选后PAP分别为(84.82±0.07)%和(99.91±0.03)%;IF检测CD133+/CD44+细胞培养后继续呈阳性表达;CD133+/CD44+细胞增殖能力高于PC-3细胞(t=11.0,P=0.008)以及高于诱导后的CD133+/CD44+细胞(t=40.1,P=0.001);CD133+/CD44+细胞经过转化生长因子-β诱导后IHC和Western blot检测PAP表达呈阳性,而未诱导的CD133+/CD44+细胞表达呈阴性.结论:MACS从PC-3细胞株中分选的CD 133+/CD44+细胞经过初步功能性鉴定具有干细胞的某些特性,可为前列腺癌干细胞的进一步探索充当铺垫.

  13. Spike sorting for polytrodes: a divide and conquer approach

    Directory of Open Access Journals (Sweden)

    Nicholas V. Swindale

    2014-02-01

    Full Text Available In order to determine patterns of neural activity, spike signals recorded by extracellular electrodes have to be clustered (sorted with the aim of ensuring that each cluster represents all the spikes generated by an individual neuron. Many methods for spike sorting have been proposed but few are easily applicable to recordings from polytrodes which may have 16 or more recording sites. As with tetrodes, these are spaced sufficiently closely that signals from single neurons will usually be recorded on several adjacent sites. Although this offers a better chance of distinguishing neurons with similarly shaped spikes, sorting is difficult in such cases because of the high dimensionality of the space in which the signals must be classified. This report details a method for spike sorting based on a divide and conquer approach. Clusters are initially formed by assigning each event to the channel on which it is largest. Each channel-based cluster is then sub-divided into as many distinct clusters as possible. These are then recombined on the basis of pairwise tests into a final set of clusters. Pairwise tests are also performed to establish how distinct each cluster is from the others. A modified gradient ascent clustering (GAC algorithm is used to do the clustering. The method can sort spikes with minimal user input in times comparable to real time for recordings lasting up to 45 minutes. Our results illustrate some of the difficulties inherent in spike sorting, including changes in spike shape over time. We show that some physiologically distinct units may have very similar spike shapes. We show that RMS measures of spike shape similarity are not sensitive enough to discriminate clusters that can otherwise be separated by principal components analysis. Hence spike sorting based on least-squares matching to templates may be unreliable. Our methods should be applicable to tetrodes and scaleable to larger multi-electrode arrays (MEAs.

  14. Mechanically robust microfluidics and bulk wave acoustics to sort microparticles

    Science.gov (United States)

    Dauson, Erin R.; Gregory, Kelvin B.; Greve, David W.; Healy, Gregory P.; Oppenheim, Irving J.

    2016-04-01

    Sorting microparticles (or cells, or bacteria) is significant for scientific, medical and industrial purposes. Research groups have used lithium niobate SAW devices to produce standing waves, and then to align microparticles at the node lines in polydimethylsiloxane (PDMS, silicone) microfluidic channels. The "tilted angle" (skewed) configuration is a recent breakthrough producing particle trajectories that cross multiple node lines, making it practical to sort particles. However, lithium niobate wafers and PDMS microfluidic channels are not mechanically robust. We demonstrate "tilted angle" microparticle sorting in novel devices that are robust, rapidly prototyped, and manufacturable. We form our microfluidic system in a rigid polymethyl methacrylate (PMMA, acrylic) prism, sandwiched by lead-zirconium-titanate (PZT) wafers, operating in through-thickness mode with inertial backing, that produce standing bulk waves. The overall configuration is compact and mechanically robust, and actuating PZT wafers in through-thickness mode is highly efficient. Moving to this novel configuration introduced new acoustics questions involving internal reflections, but we show experimental images confirming the intended nodal geometry. Microparticles in "tilted angle" devices display undulating trajectories, where deviation from the straight path increases with particle diameter and with excitation voltage to create the mechanism by which particles are sorted. We show a simplified analytical model by which a "phase space" is constructed to characterize effective particle sorting, and we compare our experimental data to the predictions from that simplified model; precise correlation is not expected and is not observed, but the important physical trends from the model are paralleled in the measured particle trajectories.

  15. A microfluidic device to sort capsules by deformability

    CERN Document Server

    Zhu, L; Mitra, Dhrubaditya; Brandt, Luca

    2014-01-01

    Guided by extensive numerical simulations, we propose a microfluidic device that can sort elastic capsules by their deformability. The device consists of a duct embedded with a semi-cylindrical obstacle, and a diffuser which further enhances the sorting capability. We demonstrate that the device can operate reasonably well under changes in the initial position of the the capsule. The efficiency of the device remains essentially unaltered under small changes of the obstacle shape (from semi-circular to semi-elliptic cross-section). Confinement along the direction perpendicular to the plane of the device increases its efficiency. This work is the first numerical study of cell sorting by a realistic microfluidic device.

  16. "A Shock of Electricity Just Sort of Goes through My Body": Physical Activity and Embodied Reflexive Practices in Young Female Ballet Dancers

    Science.gov (United States)

    Wellard, Ian; Pickard, Angela; Bailey, Richard

    2007-01-01

    Participation in physical activities, in and out of school, remains heavily influenced by social constructions of gendered behaviour. In addition, the body plays a significant part in the presentation of legitimate performances of physical practice and the construction of a physical "identity". The consequence is that in formalized…

  17. Sorting and Selection in Posets

    CERN Document Server

    Daskalakis, Constantinos; Mossel, Elchanan; Riesenfeld, Samantha; Verbin, Elad

    2007-01-01

    Classical problems of sorting and searching assume an underlying linear ordering of the objects being compared. In this paper, we study a more general setting, in which some pairs of objects are incomparable. This generalization is relevant in applications related to rankings in sports, college admissions, or conference submissions. It also has potential applications in biology, such as comparing the evolutionary fitness of different strains of bacteria, or understanding input-output relations among a set of metabolic reactions or the causal influences among a set of interacting genes or proteins. Our results improve and extend results from two decades ago of Faigle and Tur\\'{a}n. A measure of complexity of a partially ordered set (poset) is its width. Our algorithms obtain information about a poset by queries that compare two elements. We present an algorithm that sorts, i.e. completely identifies, a width w poset of size n and has query complexity O(wn + nlog(n)), which is within a constant factor of the in...

  18. 血小板微颗粒的促凝功能与流式细胞术绝对计数值的相关性%Correlation between the coagulation function of the platelet-derived mi-crovesicle and the absolute count of flow cytometry cell sorting

    Institute of Scientific and Technical Information of China (English)

    饶冬东; 张福辉; 薛晓光; 邱君

    2015-01-01

    目的:探讨流式细胞术所获得血小板微颗粒计数与其功能间的关系。方法选取本院2012年9月~2014年9月的100例健康孕产妇及患有合并症孕产妇患者的血液样本作为研究对象,经流式细胞术计数分析及三种功能分析,探讨其相关性。结果流式细胞术获得的乳黏素蛋白促凝血的微粒体计数与Zymuphen MP活性呈弱相关(r越0.5370,P<0.01);与内在凝血酶潜力ETP呈正相关(r越0.7444,P<0.01);与STA磷脂(PPL)促凝分析呈负相关(r=-0.7872,P<0.01)。膜联蛋白V+及促凝血的血小板源性微颗粒的含量水平与功能分析一致。结论血小板微颗粒的促凝功能与流式细胞术绝对计数值密切相关,多参数的使用将会提供更多的生物学信息。%Objective To explore the relationship between the number of platelet-derived microvesicle sorted by flow cytometry cell sorting and their function. Methods 100 copies of blood samplesobtained from healthy maternal women or maternal women with complications from September 2012 to September 2014 in our hospital were selected as the re-search object.The number of platelet microvesicles were calculated by flow cytometry cell sorting,and their functions were recorded by three function analysis.Their relationship was explored. Results The number of platelet microvesicles was slightly correlated with Zymuphen MP activity (r=0.5370,P<0.01) and positively correlated with ETP (r=0.7444,P<0.01),while negatively correlated with STA PPL (r=-0.7872,P<0.01).Membrane associated protein V+was related to the number of coagulation of platelet microvesicles,which was helpful for function analysis. Conclusion The coagulation of platelet microvesicles is closely related to their number counted by flow cytometry cell sorting and the application of multiple parameters will provide valuable biological information.

  19. Fixing the Sorting Algorithm for Android, Java and Python

    NARCIS (Netherlands)

    C.P.T. de Gouw (Stijn); F.S. de Boer (Frank)

    2015-01-01

    htmlabstractTim Peters developed the Timsort hybrid sorting algorithm in 2002. TimSort was first developed for Python, a popular programming language, but later ported to Java (where it appears as java.util.Collections.sort and java.util.Arrays.sort). TimSort is today used as the default sorting alg

  20. Fixing the Sorting Algorithm for Android, Java and Python

    NARCIS (Netherlands)

    Gouw, C.P.T. de; Boer, F.S. de

    2015-01-01

    Tim Peters developed the Timsort hybrid sorting algorithm in 2002. TimSort was first developed for Python, a popular programming language, but later ported to Java (where it appears as java.util.Collections.sort and java.util.Arrays.sort). TimSort is today used as the default sorting algorithm in Ja

  1. Fixing the Sorting Algorithm for Android, Java and Python

    NARCIS (Netherlands)

    C.P.T. de Gouw (Stijn); F.S. de Boer (Frank)

    2015-01-01

    htmlabstractTim Peters developed the Timsort hybrid sorting algorithm in 2002. TimSort was first developed for Python, a popular programming language, but later ported to Java (where it appears as java.util.Collections.sort and java.util.Arrays.sort). TimSort is today used as the default sorting

  2. Activity-Dependent Ubiquitination of GluA1 and GluA2 Regulates AMPA Receptor Intracellular Sorting and Degradation

    OpenAIRE

    Jocelyn Widagdo; Ye Jin Chai; Margreet C. Ridder; Yu Qian Chau; Richard C. Johnson; Pankaj Sah; Richard L. Huganir; Victor Anggono

    2015-01-01

    AMPA receptors (AMPARs) have recently been shown to undergo post-translational ubiquitination in mammalian neurons. However, the underlying molecular mechanisms are poorly understood and remain controversial. Here, we report that all four AMPAR subunits (GluA1-4) are rapidly ubiquitinated upon brief application of AMPA or bicuculline in cultured neurons. This process is Ca2+ dependent and requires the activity of L-type voltage-gated Ca2+ channels and Ca2+/calmodulin-dependent kinase II. The ...

  3. Energy efficient data sorting using standard sorting algorithms

    KAUST Repository

    Bunse, Christian

    2011-01-01

    Protecting the environment by saving energy and thus reducing carbon dioxide emissions is one of todays hottest and most challenging topics. Although the perspective for reducing energy consumption, from ecological and business perspectives is clear, from a technological point of view, the realization especially for mobile systems still falls behind expectations. Novel strategies that allow (software) systems to dynamically adapt themselves at runtime can be effectively used to reduce energy consumption. This paper presents a case study that examines the impact of using an energy management component that dynamically selects and applies the "optimal" sorting algorithm, from an energy perspective, during multi-party mobile communication. Interestingly, the results indicate that algorithmic performance is not key and that dynamically switching algorithms at runtime does have a significant impact on energy consumption. © Springer-Verlag Berlin Heidelberg 2011.

  4. Cell division activity during apical hook development

    NARCIS (Netherlands)

    Raz, V.; Koornneef, M.

    2001-01-01

    Growth during plant development is predominantly governed by the combined activities of cell division and cell elongation. The relative contribution of both activities controls the growth of a tissue. A fast change in growth is exhibited at the apical hypocotyl of etiolated seedlings where cells

  5. Cell division activity during apical hook development

    NARCIS (Netherlands)

    Raz, V.; Koornneef, M.

    2001-01-01

    Growth during plant development is predominantly governed by the combined activities of cell division and cell elongation. The relative contribution of both activities controls the growth of a tissue. A fast change in growth is exhibited at the apical hypocotyl of etiolated seedlings where cells gro

  6. Receptorligand sorting along the endocytic pathway

    CERN Document Server

    Linderman, Jennifer J

    1989-01-01

    This research monograph focuses on a biomolecular separation process that occurs within most cells. Two types of molecules, receptors and ligands, are separated and routed along different intracellular pathways; this is a critical step in the process of receptor-mediated endocytosis. The development of an understanding of the basic mechanisms of this separation process is presented, with an emphasis on discovering the fundamental and measurable parameters that influence the event. Mathematical models of sorting are evaluated to predict the range of possible outcomes. These are compared with a variety of experimental data on different receptor/ligand systems. In addition, the influence of the separation on overall receptor/ligand processing dynamics is discussed. The book is intended for both biomathematicians and biologists. It is not necessary to understand the details of the model equations and their solution in order to test the models experimentally. The analysis suggests experiments that might be done to...

  7. Viral Evasion of Natural Killer Cell Activation

    OpenAIRE

    Yi Ma; Xiaojuan Li; Ersheng Kuang

    2016-01-01

    Natural killer (NK) cells play a key role in antiviral innate defenses because of their abilities to kill infected cells and secrete regulatory cytokines. Additionally, NK cells exhibit adaptive memory-like antigen-specific responses, which represent a novel antiviral NK cell defense mechanism. Viruses have evolved various strategies to evade the recognition and destruction by NK cells through the downregulation of the NK cell activating receptors. Here, we review the recent findings on viral...

  8. The method of sorting out perivascular stem cells from human adipose tissue through flow cytometry%流式分析人脂肪组织中血管周围干细胞含量的方法探究

    Institute of Scientific and Technical Information of China (English)

    徐峰; 刘舒云; 王鑫; 彭江; 卢世璧; 袁玫; 许文静; 郭全义

    2015-01-01

    目的建立人脂肪组织中分离血管周围干细胞(PSCs)的方法,并研究其在脂肪组织细胞中所占的比例,为血管周围干细胞作为骨和软骨组织工程新的种子细胞奠定基础。  方法取人的脂肪组织分别用 I 型胶原酶和 II 型胶原酶消化得到血管基质成分(SVF),用细胞计数仪及流式细胞仪检测 SVF 中细胞密度、活细胞比例和 PSCs 细胞所占的比例。  结果用细胞计数仪分析得出用 II 型胶原酶消化脂肪组织所得到的 SVF 中活细胞比例更高,且差异具有统计学意义(P  结论使用 II 型胶原酶消化脂肪组织可以得到更多的血管周围干细胞 PSCs,其在脂肪组织中的含量可以满足骨和软骨损伤后自体细胞移植修复的需要。%Objective To establish the method of sorting out perivascular stem cells (PSCs) from human adipose tissue and study the proportion of these cells in adipose tissue cells. This research is to explore new seed cells for the bone and cartilage tissue engineering. Methods Stromal vascular fraction (SVF) was got from human adipose tissue that was digested by collagenase type I or collagenase type II. The cell density, proportion of living cells and proportion of PSCs in SVF were tested by the cell count and flow cytometry (FCM). Results The proportion of living cells in SVF digested by collagenase type II was much higher through analyzing by the cell count and the difference was statistically significant (P Conclusion A higher amount of PSCs can be got from human adipose digested by collagenase type II, and the content of PSCs in the adipose tissue can satisfy the needs of autologous cell transplantation for the bone and cartilage repair.

  9. Mast cells enhance T cell activation: Importance of mast cell-derived TNF

    Science.gov (United States)

    Nakae, Susumu; Suto, Hajime; Kakurai, Maki; Sedgwick, Jonathon D.; Tsai, Mindy; Galli, Stephen J.

    2005-05-01

    Mast cells are not only important effector cells in immediate hypersensitivity reactions and immune responses to pathogens but also can contribute to T cell-mediated disorders. However, the mechanisms by which mast cells might influence T cells in such settings are not fully understood. We find that mast cells can enhance proliferation and cytokine production in multiple T cell subsets. Mast cell-dependent enhancement of T cell activation can be promoted by FcRI-dependent mast cell activation, TNF production by both mast cells and T cells, and mast cell-T cell contact. However, at high concentrations of cells, mast cells can promote T cell activation independent of IgE or TNF. Finally, mast cells also can promote T cell activation by means of soluble factors. These findings identify multiple mechanisms by which mast cells can influence T cell proliferation and cytokine production. allergy | asthma | autoimmunity | cytokines | immune response

  10. Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing

    Science.gov (United States)

    Bolognesi, Chiara; Forcato, Claudio; Buson, Genny; Fontana, Francesca; Mangano, Chiara; Doffini, Anna; Sero, Valeria; Lanzellotto, Rossana; Signorini, Giulio; Calanca, Alex; Sergio, Maximilian; Romano, Rita; Gianni, Stefano; Medoro, Gianni; Giorgini, Giuseppe; Morreau, Hans; Barberis, Massimo; Corver, Willem E.; Manaresi, Nicolò

    2016-01-01

    Precision medicine in oncology requires an accurate characterization of a tumor molecular profile for patient stratification. Though targeted deep sequencing is an effective tool to detect the presence of somatic sequence variants, a significant number of patient specimens do not meet the requirements needed for routine clinical application. Analysis is hindered by contamination of normal cells and inherent tumor heterogeneity, compounded with challenges of dealing with minute amounts of tissue and DNA damages common in formalin-fixed paraffin-embedded (FFPE) specimens. Here we present an innovative workflow using DEPArray™ system, a microchip-based digital sorter to achieve 100%-pure, homogenous subpopulations of cells from FFPE samples. Cells are distinguished by fluorescently labeled antibodies and DNA content. The ability to address tumor heterogeneity enables unambiguous determination of true-positive sequence variants, loss-of-heterozygosity as well as copy number variants. The proposed strategy overcomes the inherent trade-offs made between sensitivity and specificity in detecting genetic variants from a mixed population, thus rescuing for analysis even the smaller clinical samples with low tumor cellularity. PMID:26864208

  11. Learning sorting algorithms through visualization construction

    Science.gov (United States)

    Cetin, Ibrahim; Andrews-Larson, Christine

    2016-01-01

    Recent increased interest in computational thinking poses an important question to researchers: What are the best ways to teach fundamental computing concepts to students? Visualization is suggested as one way of supporting student learning. This mixed-method study aimed to (i) examine the effect of instruction in which students constructed visualizations on students' programming achievement and students' attitudes toward computer programming, and (ii) explore how this kind of instruction supports students' learning according to their self-reported experiences in the course. The study was conducted with 58 pre-service teachers who were enrolled in their second programming class. They expect to teach information technology and computing-related courses at the primary and secondary levels. An embedded experimental model was utilized as a research design. Students in the experimental group were given instruction that required students to construct visualizations related to sorting, whereas students in the control group viewed pre-made visualizations. After the instructional intervention, eight students from each group were selected for semi-structured interviews. The results showed that the intervention based on visualization construction resulted in significantly better acquisition of sorting concepts. However, there was no significant difference between the groups with respect to students' attitudes toward computer programming. Qualitative data analysis indicated that students in the experimental group constructed necessary abstractions through their engagement in visualization construction activities. The authors of this study argue that the students' active engagement in the visualization construction activities explains only one side of students' success. The other side can be explained through the instructional approach, constructionism in this case, used to design instruction. The conclusions and implications of this study can be used by researchers and

  12. HERV-K activation is strictly required to sustain CD133+ melanoma cells with stemness features.

    Science.gov (United States)

    Argaw-Denboba, Ayele; Balestrieri, Emanuela; Serafino, Annalucia; Cipriani, Chiara; Bucci, Ilaria; Sorrentino, Roberta; Sciamanna, Ilaria; Gambacurta, Alessandra; Sinibaldi-Vallebona, Paola; Matteucci, Claudia

    2017-01-26

    Melanoma is a heterogeneous tumor in which phenotype-switching and CD133 marker have been associated with metastasis promotion and chemotherapy resistance. CD133 positive (CD133+) subpopulation has also been suggested as putative cancer stem cell (CSC) of melanoma tumor. Human endogenous retrovirus type K (HERV-K) has been described to be aberrantly activated during melanoma progression and implicated in the etiopathogenesis of disease. Earlier, we reported that stress-induced HERV-K activation promotes cell malignant transformation and reduces the immunogenicity of melanoma cells. Herein, we investigated the correlation between HERV-K and the CD133+ melanoma cells during microenvironmental modifications. TVM-A12 cell line, isolated in our laboratory from a primary human melanoma lesion, and other commercial melanoma cell lines (G-361, WM-115, WM-266-4 and A375) were grown and maintained in the standard and stem cell media. RNA interference, Real-time PCR, flow cytometry analysis, self-renewal and migration/invasion assays were performed to characterize cell behavior and HERV-K expression. Melanoma cells, exposed to stem cell media, undergo phenotype-switching and expansion of CD133+ melanoma cells, concomitantly promoted by HERV-K activation. Notably, the sorted CD133+ subpopulation showed stemness features, characterized by higher self-renewal ability, embryonic genes expression, migration and invasion capacities compared to the parental cell line. RNA interference-mediated downregulation experiments showed that HERV-K has a decisive role to expand and maintain the CD133+ melanoma subpopulation during microenvironmental modifications. Similarly, non nucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz and nevirapine were effective to restrain the activation of HERV-K in melanoma cells, to antagonize CD133+ subpopulation expansion and to induce selective high level apoptosis in CD133+ cells. HERV-K activation promotes melanoma cells phenotype

  13. Potent Anti-HIV Chemokine Analogs Direct Post-Endocytic Sorting of CCR5.

    Directory of Open Access Journals (Sweden)

    Claudia Bönsch

    Full Text Available G protein-coupled receptors (GPCRs are desensitized and internalized following activation. They are then subjected to post-endocytic sorting (degradation, slow recycling or fast recycling. The majority of research on post-endocytic sorting has focused on the role of sequence-encoded address structures on receptors. This study focuses on trafficking of CCR5, a GPCR chemokine receptor and the principal entry coreceptor for HIV. Using Chinese Hamster Ovary cells stably expressing CCR5 we show that two different anti-HIV chemokine analogs, PSC-RANTES and 5P14-RANTES, direct receptor trafficking into two distinct subcellular compartments: the trans-Golgi network and the endosome recycling compartment, respectively. Our results indicate that a likely mechanism for ligand-directed sorting of CCR5 involves capacity of the chemokine analogs to elicit the formation of durable complexes of CCR5 and arrestin2 (beta-arrestin-1, with PSC-RANTES eliciting durable association in contrast to 5P14-RANTES, which elicits only transient association.

  14. On the Construction of Sorted Reactive Systems

    DEFF Research Database (Denmark)

    Birkedal, Lars; Debois, Søren; Hildebrandt, Thomas

    2008-01-01

    We develop a theory of sorted bigraphical reactive systems. Every application of bigraphs in the literature has required an extension, a sorting, of pure bigraphs. In turn, every such application has required a redevelopment of the theory of pure bigraphical reactive systems for the sorting at hand...... bigraphs. Technically, we give our construction for ordinary reactive systems, then lift it to bigraphical reactive systems. As such, we give also a construction of sortings for ordinary reactive systems. This construction is an improvement over previous attempts in that it produces smaller and much more...

  15. Ubiquitination-deubiquitination balance dictates ligand-stimulated PTHR sorting.

    Science.gov (United States)

    Alonso, Verónica; Magyar, Clara E; Wang, Bin; Bisello, Alessandro; Friedman, Peter A

    2011-12-01

    Parathyroid hormone receptors (PTHR) are promptly internalized upon stimulation by activating (PTH[1-84], PTH[1-34]) and non-activating (PTH[7-84], PTH[7-34]) ligands. Here, we characterized the mechanism regulating the sorting of internalized receptors between recycling and degradative pathways. PTHR recycles faster after challenge with PTH(1-34) than with PTH(7-34). PTHR recycling is complete by 2 h after PTH(1-34) stimulation, but incomplete at this time in cells treated with PTH(7-34). The slower and incomplete recycling induced by PTH(7-34) is due to proteasomal degradation. Both PTH(1-34) and PTH(7-34) induced PTHR polyubiquitination. Ubiquitination by PTH(1-34) was transient, whereas receptor ubiquitination after PTH(7-34) was sustained. PTH(1-34), but not PTH(7-34), induced expression of the PTHR-specific deubiquitinating enzyme USP2. Overexpression of USP2 prevented PTH(7-34)-induced PTHR degradation. We conclude that PTH(1-34) promotes coupled PTHR ubiquitination and deubiquitination, whereas PTH(7-34) activates only ubiquitination, thereby leading to PTHR downregulation. These findings may explain PTH resistance in diseases associated with elevated PTH(7-84) levels. Copyright © 2011 American Society for Bone and Mineral Research.

  16. Ubiquitination–Deubiquitination Balance Dictates Ligand-stimulated PTHR Sorting

    Science.gov (United States)

    Alonso, Verónica; Magyar, Clara E.; Wang, Bin; Bisello, Alessandro; Friedman, Peter A.

    2011-01-01

    Parathyroid hormone receptors (PTHR) are promptly internalized upon stimulation by activating [PTH(1–84), PTH(1–34)] and non-activating [PTH(7–84), PTH(7–34)] ligands. Here, we characterized the mechanism regulating the sorting of internalized receptors between recycling and degradative pathways. PTHR recycles faster after challenge with PTH(1–34) than with PTH(7–34). PTHR recycling is complete by 2 hr after PTH(1–34) stimulation but incomplete at this time in cells treated with PTH(7–34). The slower and incomplete recycling induced by PTH(7–34) is due to proteasomal degradation. Both PTH(1–34) and PTH(7–34) induced PTHR polyubiquitination. Ubiquitination by PTH(1–34) was transient, whereas receptor ubiquitination following PTH(7–34) was sustained. PTH(1–34), but not PTH(7–34), induced expression of the PTHR-specific deubiquitinating enzyme USP2. Overexpression of USP2 prevented PTH(7–34)-induced PTHR degradation. We conclude that PTH(1–34) promotes coupled PTHR ubiquitination and deubiquitination, whereas PTH(7–34) activates only ubiquitination, thereby leading to PTHR downregulation. These findings may explain PTH resistance in diseases associated with elevated PTH(7–84) levels. PMID:21898592

  17. Flow cytometric sorting of fecal bacteria after in situ hybridization with polynucleotide probes.

    Science.gov (United States)

    Bruder, Lena M; Dörkes, Marcel; Fuchs, Bernhard M; Ludwig, Wolfgang; Liebl, Wolfgang

    2016-10-01

    The gut microbiome represents a key contributor to human physiology, metabolism, immune function, and nutrition. Elucidating the composition and genetics of the gut microbiota under various conditions is essential to understand how microbes function individually and as a community. Metagenomic analyses are increasingly used to study intestinal microbiota. However, for certain scientific questions it is sufficient to examine taxon-specific submetagenomes, covering selected bacterial genera in a targeted manner. Here we established a new variant of fluorescence in situ hybridization (FISH) combined with fluorescence-activated cell sorting (FACS), providing access to the genomes of specific taxa belonging to the complex community of the intestinal microbiota. In contrast to standard oligonucleotide probes, the RNA polynucleotide probe used here, which targets domain III of the 23S rRNA gene, extends the resolution power in environmental samples by increasing signal intensity. Furthermore, cells hybridized with the polynucleotide probe are not subjected to harsh pretreatments, and their genetic information remains intact. The protocol described here was tested on genus-specifically labeled cells in various samples, including complex fecal samples from different laboratory mouse types that harbor diverse intestinal microbiota. Specifically, as an example for the protocol described here, RNA polynucleotide probes could be used to label Enterococcus cells for subsequent sorting by flow cytometry. To detect and quantify enterococci in fecal samples prior to enrichment, taxon-specific PCR and qPCR detection systems have been developed. The accessibility of the genomes from taxon-specifically sorted cells for subsequent molecular analyses was demonstrated by amplification of functional genes. Copyright © 2016 Elsevier GmbH. All rights reserved.

  18. Mast cell activation syndromes presenting as anaphylaxis.

    Science.gov (United States)

    Akin, Cem

    2015-05-01

    Anaphylaxis results from severe systemic mast cell activation. In addition to IgE-mediated and physical triggers, it may occur with a clonal mast cell disease and in an idiopathic fashion without clear provoking factors. Disorders of mast cell activation are classified into primary (clonal), secondary, and idiopathic. Mast cell activation syndrome (MCAS) is a multisystem disorder characterized by objective documentation of elevated mast cell mediators during attacks and a favorable response to antimediator therapy. It should be considered in the differential diagnosis of patients presenting with recurrent anaphylaxis without a clear cause. This article discusses the diagnosis of MCAS.

  19. ARF6 and GASP-1 are post-endocytic sorting proteins selectively involved in the intracellular trafficking of dopamine D₂ receptors mediated by GRK and PKC in transfected cells.

    Science.gov (United States)

    Cho, D I; Zheng, M; Min, C; Kwon, K J; Shin, C Y; Choi, H K; Kim, K M

    2013-03-01

    GPCRs undergo both homologous and heterologous regulatory processes in which receptor phosphorylation plays a critical role. The protein kinases responsible for each pathway are well established; however, other molecular details that characterize each pathway remain unclear. In this study, the molecular mechanisms that determine the differences in the functional roles and intracellular trafficking between homologous and PKC-mediated heterologous internalization pathways for the dopamine D₂ receptor were investigated. All of the S/T residues located within the intracellular loops of D₂ receptor were mutated, and the residues responsible for GRK- and PKC-mediated internalization were determined in HEK-293 cells and SH-SY5Y cells. The functional role of receptor internalization and the cellular components that determine the post-endocytic fate of internalized D₂ receptors were investigated in the transfected cells. T134, T225/S228/S229 and S325 were involved in PKC-mediated D₂ receptor desensitization. S229 and adjacent S/T residues mediated the PKC-dependent internalization of D₂ receptors, which induced down-regulation and desensitization. S/T residues within the second intracellular loop and T225 were the major residues involved in GRK-mediated internalization of D₂ receptors, which induced receptor resensitization. ARF6 mediated the recycling of D₂ receptors internalized in response to agonist stimulation. In contrast, GASP-1 mediated the down-regulation of D₂ receptors internalized in a PKC-dependent manner. GRK- and PKC-mediated internalizations of D₂ receptors occur through different intracellular trafficking pathways and mediate distinct functional roles. Distinct S/T residues within D₂ receptors and different sorting proteins are involved in the dissimilar regulation of D₂ receptors by GRK2 and PKC. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

  20. ARF6 and GASP-1 are post-endocytic sorting proteins selectively involved in the intracellular trafficking of dopamine D2 receptors mediated by GRK and PKC in transfected cells

    Science.gov (United States)

    Cho, DI; Zheng, M; Min, C; Kwon, KJ; Shin, CY; Choi, HK; Kim, KM

    2013-01-01

    Background and Purpose GPCRs undergo both homologous and heterologous regulatory processes in which receptor phosphorylation plays a critical role. The protein kinases responsible for each pathway are well established; however, other molecular details that characterize each pathway remain unclear. In this study, the molecular mechanisms that determine the differences in the functional roles and intracellular trafficking between homologous and PKC-mediated heterologous internalization pathways for the dopamine D2 receptor were investigated. Experimental Approach All of the S/T residues located within the intracellular loops of D2 receptor were mutated, and the residues responsible for GRK- and PKC-mediated internalization were determined in HEK-293 cells and SH-SY5Y cells. The functional role of receptor internalization and the cellular components that determine the post-endocytic fate of internalized D2 receptors were investigated in the transfected cells. Key Results T134, T225/S228/S229 and S325 were involved in PKC-mediated D2 receptor desensitization. S229 and adjacent S/T residues mediated the PKC-dependent internalization of D2 receptors, which induced down-regulation and desensitization. S/T residues within the second intracellular loop and T225 were the major residues involved in GRK-mediated internalization of D2 receptors, which induced receptor resensitization. ARF6 mediated the recycling of D2 receptors internalized in response to agonist stimulation. In contrast, GASP-1 mediated the down-regulation of D2 receptors internalized in a PKC-dependent manner. Conclusions and Implications GRK- and PKC-mediated internalizations of D2 receptors occur through different intracellular trafficking pathways and mediate distinct functional roles. Distinct S/T residues within D2 receptors and different sorting proteins are involved in the dissimilar regulation of D2 receptors by GRK2 and PKC. PMID:23082996

  1. Design and realization of sort manipulator of crystal-angle sort machine

    Science.gov (United States)

    Wang, Ming-shun; Chen, Shu-ping; Guan, Shou-ping; Zhang, Yao-wei

    2005-12-01

    It is a current tendency of development in automation technology to replace manpower with manipulators in working places where dangerous, harmful, heavy or repetitive work is involved. The sort manipulator is installed in a crystal-angle sort machine to take the place of manpower, and engaged in unloading and sorting work. It is the outcome of combing together mechanism, electric transmission, and pneumatic element and micro-controller control. The step motor makes the sort manipulator operate precisely. The pneumatic elements make the sort manipulator be cleverer. Micro-controller's software bestows some simple artificial intelligence on the sort manipulator, so that it can precisely repeat its unloading and sorting work. The combination of manipulator's zero position and step motor counting control puts an end to accumulating error in long time operation. A sort manipulator's design in the practice engineering has been proved to be correct and reliable.

  2. Choreography of MAGUKs during T cell activation.

    Science.gov (United States)

    Rincón, Mercedes; Davis, Roger J

    2007-02-01

    T cell receptor activation requires the membrane-associated guanylate kinase CARMA1. A new study finds that a second such kinase, Dlgh1, is also required specifically for activation of the alternative p38 kinase pathway.

  3. Active cell mechanics: Measurement and theory.

    Science.gov (United States)

    Ahmed, Wylie W; Fodor, Étienne; Betz, Timo

    2015-11-01

    Living cells are active mechanical systems that are able to generate forces. Their structure and shape are primarily determined by biopolymer filaments and molecular motors that form the cytoskeleton. Active force generation requires constant consumption of energy to maintain the nonequilibrium activity to drive organization and transport processes necessary for their function. To understand this activity it is necessary to develop new approaches to probe the underlying physical processes. Active cell mechanics incorporates active molecular-scale force generation into the traditional framework of mechanics of materials. This review highlights recent experimental and theoretical developments towards understanding active cell mechanics. We focus primarily on intracellular mechanical measurements and theoretical advances utilizing the Langevin framework. These developing approaches allow a quantitative understanding of nonequilibrium mechanical activity in living cells. This article is part of a Special Issue entitled: Mechanobiology.

  4. Cell death sensitization of leukemia cells by opioid receptor activation

    Science.gov (United States)

    Friesen, Claudia; Roscher, Mareike; Hormann, Inis; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf A.; Debatin, Klaus-Michael; Miltner, Erich

    2013-01-01

    Cyclic AMP (cAMP) regulates a number of cellular processes and modulates cell death induction. cAMP levels are altered upon stimulation of specific G-protein-coupled receptors inhibiting or activating adenylyl cyclases. Opioid receptor stimulation can activate inhibitory Gi-proteins which in turn block adenylyl cyclase activity reducing cAMP. Opioids such as D,L-methadone induce cell death in leukemia cells. However, the mechanism how opioids trigger apoptosis and activate caspases in leukemia cells is not understood. In this study, we demonstrate that downregulation of cAMP induced by opioid receptor activation using the opioid D,L-methadone kills and sensitizes leukemia cells for doxorubicin treatment. Enhancing cAMP levels by blocking opioid-receptor signaling strongly reduced D,L-methadone-induced apoptosis, caspase activation and doxorubicin-sensitivity. Induction of cell death in leukemia cells by activation of opioid receptors using the opioid D,L-methadone depends on critical levels of opioid receptor expression on the cell surface. Doxorubicin increased opioid receptor expression in leukemia cells. In addition, the opioid D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux in leukemia cells, suggesting that the opioid D,L-methadone as well as doxorubicin mutually increase their cytotoxic potential. Furthermore, we found that opioid receptor activation using D,L-methadone alone or in addition to doxorubicin inhibits tumor growth significantly in vivo. These results demonstrate that opioid receptor activation via triggering the downregulation of cAMP induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment. Hence, opioid receptor activation seems to be a promising strategy to improve anticancer therapies. PMID:23633472

  5. Automated multi-parametric sorting of micron-sized particles via multi-trap laser tweezers

    Science.gov (United States)

    Kaputa, Daniel S.

    The capabilities of laser tweezers have rapidly expanded since the first demonstration by Ashkin and co-workers in 1970 of the ability to trap particles using optical energy. Laser tweezers have been used to measure piconewton forces in many biological and material science application, sort bacteria, measure DNA bond strength, and even perform microsurgery. The laser tweezers system developed for this dissertation foreshadows the next generation of laser tweezer systems that provide automated particle sorted based upon multiple criteria. Many laser tweezer sorting applications today entail the operator sorting cells from a bulk sample, one by one. This dissertation demonstrates the technologies of pattern recognition and image processing that allow for an entire microscope slide to be sorted without any operator intervention. We already live in an automated world where the cars we drive are built by machines instead of humans. The technology is there, and the only factors limiting the advancements of fully automated biological instrumentation is the lack of developers with the appropriate knowledge sets. This dissertation introduces the concept of sorting particles via a multi-parametric approach where several parameters such as size, fluorescence, and Raman spectra are used as sorting criteria. Since the advent of laser tweezers, several groups have demonstrated the ability to sort cells and other particle by size, or by fluorescence, or by any other parameter, but to our knowledge there does not exist a laser tweezer sorting system that can sort particles based upon multiple parameters. Sorting via a single parameter can be a severe limitation as the method lacks the robustness and class specificity that exists when sorting based upon multiple parameters. Simply put, it makes more sense to determine the worth of a baseball card by considering it's condition as well as it's age, rather then solely upon its condition. By adding another parameter such as the name of

  6. Differential activation of dendritic cells by nerve growth factor and brain-derived neurotrophic factor.

    Science.gov (United States)

    Noga, O; Peiser, M; Altenähr, M; Knieling, H; Wanner, R; Hanf, G; Grosse, R; Suttorp, N

    2007-11-01

    Neurotrophins are involved in inflammatory reactions influencing several cells in health and disease including allergy and asthma. Dendritic cells (DCs) play a major role in the induction of inflammatory processes with an increasing role in allergic diseases as well. The aim of this study was to investigate the influence of neurotrophins on DC function. Monocyte-derived dendritic cells were generated from allergic and non-allergic donors. Neurotrophin receptors were demonstrated by western blotting, flow cytometry and fluorescence microscopy. Activation of small GTPases was evaluated by pull-down assays. DCs were incubated with nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and supernatants were collected for measurement of IL-4, IL-6, IL-10, IL-12p70, TNF-alpha and TGF-beta. Receptor proteins were detectable by western blot, fluorescence activated cell sorting analysis and fluorescence microscopy. Signalling after neurotrophin stimulation occurred in a ligand-specific pattern. NGF led to decreased RhoA and increased Rac activation, while BDNF affected RhoA and Rac activity in a reciprocal fashion. Cells of allergics released a significantly increased amount of IL-6, while for healthy subjects a significantly higher amount of IL-10 was found. These data indicate that DCs are activated by the neurotrophins NGF and BDNF by different pathways in a receptor-dependant manner. These cells then may initiate inflammatory responses based on allergic sensitization releasing preferred cytokines inducing tolerance or a T-helper type 2 response.

  7. Data parallel sorting for particle simulation

    Science.gov (United States)

    Dagum, Leonardo

    1992-01-01

    Sorting on a parallel architecture is a communications intensive event which can incur a high penalty in applications where it is required. In the case of particle simulation, only integer sorting is necessary, and sequential implementations easily attain the minimum performance bound of O (N) for N particles. Parallel implementations, however, have to cope with the parallel sorting problem which, in addition to incurring a heavy communications cost, can make the minimun performance bound difficult to attain. This paper demonstrates how the sorting problem in a particle simulation can be reduced to a merging problem, and describes an efficient data parallel algorithm to solve this merging problem in a particle simulation. The new algorithm is shown to be optimal under conditions usual for particle simulation, and its fieldwise implementation on the Connection Machine is analyzed in detail. The new algorithm is about four times faster than a fieldwise implementation of radix sort on the Connection Machine.

  8. Scalable, Multithreaded, Partially-in-Place Sorting

    Energy Technology Data Exchange (ETDEWEB)

    Haglin, David J.; Adolf, Robert D.; Mackey, Greg E.

    2013-05-20

    A recent trend in hardware development is producing computing systems that are stretching the number of cores and size of shared-memory beyond where most fundamental serial algorithms perform well. The expectation is that this trend will continue. So it makes sense to rethink our fundamental algorithms such as sorting. There are many situations where data that needs to be sorted will actually fit into the shared memory so applications could benefit from an efficient parallel sorting algorithm. When sorting large data (at least hundreds of Gigabytes) in a single shared memory, there are two factors that affect the algorithm choice. First, does the algorithm sort in-place? And second, does the algorithm scale well beyond tens of threads? Surprisingly, existing algorithms posses either one of these factors, but not both. We present an approach that gracefully degrades in performance as the amount of available working memory decreases relative to the size of the input.

  9. Data Sorting Using Graphics Processing Units

    Directory of Open Access Journals (Sweden)

    M. J. Mišić

    2012-06-01

    Full Text Available Graphics processing units (GPUs have been increasingly used for general-purpose computation in recent years. The GPU accelerated applications are found in both scientific and commercial domains. Sorting is considered as one of the very important operations in many applications, so its efficient implementation is essential for the overall application performance. This paper represents an effort to analyze and evaluate the implementations of the representative sorting algorithms on the graphics processing units. Three sorting algorithms (Quicksort, Merge sort, and Radix sort were evaluated on the Compute Unified Device Architecture (CUDA platform that is used to execute applications on NVIDIA graphics processing units. Algorithms were tested and evaluated using an automated test environment with input datasets of different characteristics. Finally, the results of this analysis are briefly discussed.

  10. Measurement of myeloid cell immune suppressive activity.

    Science.gov (United States)

    Dolcetti, Luigi; Peranzoni, Elisa; Bronte, Vincenzo

    2010-11-01

    This unit presents simple methods to assess the immunosuppressive properties of immunoregulatory cells of myeloid origin, such as myeloid-derived suppressor cells (MDSCs), both in vitro and in vivo. These methods are general and could be adapted to test the impact of different suppressive populations on T cell activation, proliferation, and cytotoxic activity; moreover they could be useful to assess the influence exerted on immune suppressive pathways by genetic modifications, chemical inhibitors, and drugs.

  11. Sorting carbon nanotubes for electronics.

    Science.gov (United States)

    Martel, Richard

    2008-11-25

    Because of their unique structure and composition, single-wall carbon nanotubes (SWNTs) are at the interface between molecules and crystalline solids. They also present properties that are ideal for making lightweight, inexpensive, and flexible electronics. The raw material is composed of a heterogeneous mixture of SWNTs that differ in helicity and diameter and, therefore, requires purification and separation. In a series of groundbreaking experiments, a robust process serving this purpose was developed based on SWNTs encapsulated in surfactants and water. Ultracentrifugation in a density gradient combined with surfactant mixtures provided buoyant density differences, enabling enrichment for both diameter and electronic properties. A new paper in this issue explores further the process through the hydrodynamic properties of SWNT-surfactant complexes. The study reveals that we have just begun to uncover the dynamics and properties of nanotube-surfactant interactions and highlights the potential that could be gained from a better understanding of their chemistry. The time scale of integration of carbon nanotubes into electronics applications remains unclear, but the recent developments in sorting out SWNTs paves the way for improving on the properties of network-based SWNTs.

  12. Enhancement of Selection, Bubble and Insertion Sorting Algorithm

    Directory of Open Access Journals (Sweden)

    Muhammad Farooq Umar

    2014-07-01

    Full Text Available In everyday life there is a large amount of data to arrange because sorting removes any ambiguities and make the data analysis and data processing very easy, efficient and provides with cost less effort. In this study a set of improved sorting algorithms are proposed which gives better performance and design idea. In this study five new sorting algorithms (Bi-directional Selection Sort, Bi-directional bubble sort, MIDBiDirectional Selection Sort, MIDBidirectional bubble sort and linear insertion sort are presented. Bi-directional Selection Sort and MIDBiDirectional Selection Sort are the enhancement on basic selection sort while Bidirectional bubble sort and MIDBidirectional bubble sort are the enhancement on basic bubble sort by changing the selection and swapping mechanism of data for sorting. Enhanced sorting algorithms reduced the iteration by half and quarter times respectively. Asymptotically complexities of these algorithms are reduced to O (n2/2 and O (n2/4 from O (n2. Linear insertion sort is the enhancement of insertion sort by changing the design of algorithm (convert two loops to one loop. So asymptotically this algorithm is converted to linear time complexity from quadratic complexity. These sorting algorithms are described using C. The proposed algorithms are analyzed using asymptotic analysis and also using machine-running time and compared with their basic sorting algorithms. In this study we also discuss how the performance and complexity can be improved by optimizing the code and design.

  13. Activated protein C modulates the proinflammatory activity of dendritic cells

    Directory of Open Access Journals (Sweden)

    Matsumoto T

    2015-05-01

    Full Text Available Takahiro Matsumoto,1,2* Yuki Matsushima,1* Masaaki Toda,1 Ziaurahman Roeen,1 Corina N D'Alessandro-Gabazza,1,5 Josephine A Hinneh,1 Etsuko Harada,1,3 Taro Yasuma,4 Yutaka Yano,4 Masahito Urawa,1,5 Tetsu Kobayashi,5 Osamu Taguchi,5 Esteban C Gabazza1 1Department of Immunology, Mie University Graduate School of Medicine, Tsu, Mie Prefecture, 2BONAC Corporation, BIO Factory 4F, Fukuoka, 3Iwade Research Institute of Mycology, 4Department of Endocrinology, Diabetes and Metabolism, 5Department of Pulmonary and Critical Care Medicine, Mie University Graduate School of Medicine, Tsu, Mie Prefecture, Japan *These authors contributed equally to this work Background: Previous studies have demonstrated the beneficial activity of activated protein C in allergic diseases including bronchial asthma and rhinitis. However, the exact mechanism of action of activated protein C in allergies is unclear. In this study, we hypothesized that pharmacological doses of activated protein C can modulate allergic inflammation by inhibiting dendritic cells. Materials and methods: Dendritic cells were prepared using murine bone marrow progenitor cells and human peripheral monocytes. Bronchial asthma was induced in mice that received intratracheal instillation of ovalbumin-pulsed dendritic cells. Results: Activated protein C significantly increased the differentiation of tolerogenic plasmacytoid dendritic cells and the secretion of type I interferons, but it significantly reduced lipopolysaccharide-mediated maturation and the secretion of inflammatory cytokines in myeloid dendritic cells. Activated protein C also inhibited maturation and the secretion of inflammatory cytokines in monocyte-derived dendritic cells. Activated protein C-treated dendritic cells were less effective when differentiating naïve CD4 T-cells from Th1 or Th2 cells, and the cellular effect of activated protein C was mediated by its receptors. Mice that received adoptive transfer of activated protein C

  14. Activated gammadelta T cells promote the activation of uveitogenic T cells and exacerbate EAU development.

    Science.gov (United States)

    Nian, Hong; Shao, Hui; O'Brien, Rebecca L; Born, Willi K; Kaplan, Henry J; Sun, Deming

    2011-07-29

    To determine how the activation of γδ T cells affects the generation of uveitogenic αβ T cells and the development of experimental autoimmune uveitis (EAU). γδ T cells were isolated from B6 mice immunized with the uveitogenic peptide IRBP(1-20) and αβ T cells from immunized TCR-δ(-/-) mice. Resting γδ T cells were prepared by culture of separated γδ T cells in cytokine-free medium for 3 to 5 days, when they showed downregulation of CD69 expression. Activated γδ T cells were prepared by incubating resting γδ T cells with anti-γδ TCR (GL3) for 2 days. Responder αβ T cells were cocultured with immunizing antigen and antigen-presenting cells. The numbers of antigen-specific T cells expressing IL-17 or IFN-γ were determined by intracellular staining followed by FACS analysis after stimulation, with or without the addition of purified γδ T cells. The cytokines in the culture medium were measured by ELISA. Highly enriched γδ T cells exert widely different effects on autoreactive αβ T cells in EAU, depending on the activation status of the γδ T cells. Whereas nonactivated γδ T cells had little effect on the activation of interphotoreceptor retinoid-binding protein-specific αβ T cells in vitro and in vivo, activated γδ T cells promoted the generation of uveitogenic T cells and exacerbated the development of EAU. The functional ability of γδ T cells is greatly influenced by their activation status. Activated γδ T cells exacerbate EAU through increased activation of uveitogenic T cells.

  15. Active Gel Model of Amoeboid Cell Motility

    CERN Document Server

    Callan-Jones, A C

    2013-01-01

    We develop a model of amoeboid cell motility based on active gel theory. Modeling the motile apparatus of a eukaryotic cell as a confined layer of finite length of poroelastic active gel permeated by a solvent, we first show that, due to active stress and gel turnover, an initially static and homogeneous layer can undergo a contractile-type instability to a polarized moving state in which the rear is enriched in gel polymer. This agrees qualitatively with motile cells containing an actomyosin-rich uropod at their rear. We find that the gel layer settles into a steadily moving, inhomogeneous state at long times, sustained by a balance between contractility and filament turnover. In addition, our model predicts an optimal value of the gel-susbstrate adhesion leading to maximum layer speed, in agreement with cell motility assays. The model may be relevant to motility of cells translocating in complex, confining environments that can be mimicked experimentally by cell migration through microchannels.

  16. Particle sorting using a porous membrane in a microfluidic device.

    Science.gov (United States)

    Wei, Huibin; Chueh, Bor-han; Wu, Huiling; Hall, Eric W; Li, Cheuk-wing; Schirhagl, Romana; Lin, Jin-Ming; Zare, Richard N

    2011-01-21

    Porous membranes have been fabricated based on the development of the perforated membrane mold [Y. Luo and R. N. Zare, Lab Chip, 2008, 8, 1688-1694] to create a single filter that contains multiple pore sizes ranging from 6.4 to 16.6 µm inside a monolithic three-dimensional poly(dimethylsiloxane) microfluidic structure. By overlapping two filters we are able to achieve smaller pore size openings (2.5 to 3.3 µm). This filter operates without any detectable irreversible clogging, which is achieved using a cross-flow placed in front of each filtration section. The utility of a particle-sorting device that contains this filter is demonstrated by separating polystyrene beads of different diameters with an efficiency greater than 99.9%. Additionally, we demonstrate the effectiveness of this particle-sorting device by separating whole blood samples into white blood cells and red blood cells with platelets.

  17. Selective Sorting of Cargo Proteins into Bacterial Membrane Vesicles*

    Science.gov (United States)

    Haurat, M. Florencia; Aduse-Opoku, Joseph; Rangarajan, Minnie; Dorobantu, Loredana; Gray, Murray R.; Curtis, Michael A.; Feldman, Mario F.

    2011-01-01

    In contrast to the well established multiple cellular roles of membrane vesicles in eukaryotic cell biology, outer membrane vesicles (OMV) produced via blebbing of prokaryotic membranes have frequently been regarded as cell debris or microscopy artifacts. Increasingly, however, bacterial membrane vesicles are thought to play a role in microbial virulence, although it remains to be determined whether OMV result from a directed process or from passive disintegration of the outer membrane. Here we establish that the human oral pathogen Porphyromonas gingivalis has a mechanism to selectively sort proteins into OMV, resulting in the preferential packaging of virulence factors into OMV and the exclusion of abundant outer membrane proteins from the protein cargo. Furthermore, we show a critical role for lipopolysaccharide in directing this sorting mechanism. The existence of a process to package specific virulence factors into OMV may significantly alter our current understanding of host-pathogen interactions. PMID:21056982

  18. An Unsupervised Online Spike-Sorting Framework.

    Science.gov (United States)

    Knieling, Simeon; Sridharan, Kousik S; Belardinelli, Paolo; Naros, Georgios; Weiss, Daniel; Mormann, Florian; Gharabaghi, Alireza

    2016-08-01

    Extracellular neuronal microelectrode recordings can include action potentials from multiple neurons. To separate spikes from different neurons, they can be sorted according to their shape, a procedure referred to as spike-sorting. Several algorithms have been reported to solve this task. However, when clustering outcomes are unsatisfactory, most of them are difficult to adjust to achieve the desired results. We present an online spike-sorting framework that uses feature normalization and weighting to maximize the distinctiveness between different spike shapes. Furthermore, multiple criteria are applied to either facilitate or prevent cluster fusion, thereby enabling experimenters to fine-tune the sorting process. We compare our method to established unsupervised offline (Wave_Clus (WC)) and online (OSort (OS)) algorithms by examining their performance in sorting various test datasets using two different scoring systems (AMI and the Adamos metric). Furthermore, we evaluate sorting capabilities on intra-operative recordings using established quality metrics. Compared to WC and OS, our algorithm achieved comparable or higher scores on average and produced more convincing sorting results for intra-operative datasets. Thus, the presented framework is suitable for both online and offline analysis and could substantially improve the quality of microelectrode-based data evaluation for research and clinical application.

  19. Support for designing waste sorting systems: A mini review.

    Science.gov (United States)

    Rousta, Kamran; Ordoñez, Isabel; Bolton, Kim; Dahlén, Lisa

    2017-08-01

    This article presents a mini review of research aimed at understanding material recovery from municipal solid waste. It focuses on two areas, waste sorting behaviour and collection systems, so that research on the link between these areas could be identified and evaluated. The main results presented and the methods used in the articles are categorised and appraised. The mini review reveals that most of the work that offered design guidelines for waste management systems was based on optimising technical aspects only. In contrast, most of the work that focused on user involvement did not consider developing the technical aspects of the system, but was limited to studies of user behaviour. The only clear consensus among the articles that link user involvement with the technical system is that convenient waste collection infrastructure is crucial for supporting source separation. This mini review reveals that even though the connection between sorting behaviour and technical infrastructure has been explored and described in some articles, there is still a gap when using this knowledge to design waste sorting systems. Future research in this field would benefit from being multidisciplinary and from using complementary methods, so that holistic solutions for material recirculation can be identified. It would be beneficial to actively involve users when developing sorting infrastructures, to be sure to provide a waste management system that will be properly used by them.

  20. 考虑任务排序策略的舰船建造车间虚拟制造单元动态调度%Virtual manufacturing cell dynamic scheduling of ship construction workshop considering the task sorting strategy

    Institute of Scientific and Technical Information of China (English)

    韩文民; 孙晓梅; 孔鹏; 吕洁

    2014-01-01

    为提高舰船制造系统作业调度的柔性和效率,从车间层的作业计划角度出发,研究周期驱动条件下的虚拟制造单元多阶段动态调度问题并构建了动态调度数学模型。模型中考虑了加工任务动态需求、设备加工能力、负荷平衡、同类设备有多台的情况且提出共享资源协调排序策略,以实现最大完工时间和总物料运输距离之和最小化的目标。运用改进蚁群算法与启发式规则的混合算法进行求解。通过某船厂的实际生产数据验证了虚拟单元动态调度方法的可行性和有效性。%In order to improve the flexibility and efficiency of shipbuilding production scheduling system and develop shop floor short - term plans, virtual manufacturing cell multi-period dynamic scheduling model was established in condition of cycle driving mechanism. The model incorporated parameters of the processing task dynamic demand, equipment processing capability, load balance, and similar equipments have multiple; and put forward sharing resources coordination sorting strategy. The objective is to minimize completion time and the total materials and components travelling distance incurred. A hybrid algorithm, based on the improved ant colony algorithm and heuristic rules was proposed to solve the complex scheduling problem. Actual production data proved that the proposed approach was feasible and effective.

  1. Activation of the complement cascade enhances motility of leukemic cells by downregulating expression of HO-1.

    Science.gov (United States)

    Abdelbaset-Ismail, A; Borkowska-Rzeszotek, S; Kubis, E; Bujko, K; Brzeźniakiewicz-Janus, K; Bolkun, L; Kloczko, J; Moniuszko, M; Basak, G W; Wiktor-Jedrzejczak, W; Ratajczak, M Z

    2017-02-01

    As a crucial arm of innate immunity, the complement cascade (ComC) is involved both in mobilization of normal hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood and in their homing to BM. Despite the fact that ComC cleavage fragments alone do not chemoattract normal HSPCs, we found that leukemia cell lines as well as clonogenic blasts from chronic myeloid leukemia and acute myeloid leukemia patients respond robustly to C3 and C5 cleavage fragments by chemotaxis and increased adhesion. This finding was supported by the detection of C3a and C5a receptors in cells from human malignant hematopoietic cell lines and patient blasts at the mRNA (reverse transcriptase-polymerase chain reaction) and protein level (fluorescence-activated cell sorting), and by the demonstration that these receptors respond to stimulation by C3a and C5a by phosphorylation of p42/44 and p38 mitogen-activated protein kinases (MAPK), and protein kinase B (PKB/AKT). We also found that inducible heme oxygenase 1 (HO-1) is a negative regulator of ComC-mediated trafficking of leukemic cells, and that stimulation of leukemic cells by C3 or C5 cleavage fragments activates p38 MAPK, which downregulates HO-1 expression, rendering cells more mobile. We conclude that activation of the ComC in leukemia/lymphoma patients (for example, as a result of accompanying infections) enhances the motility of malignant cells and contributes to their spread in a p38 MAPK-HO-1-dependent manner. Therefore, inhibition of p38 MAPK or upregulation of HO-1 by small-molecule modulators would have a beneficial effect on ameliorating cell migration-mediated expansion of leukemia/lymphoma cells when the ComC becomes activated.

  2. Altered differentiation and paracrine stimulation of mammary epithelial cell proliferation by conditionally activated Smoothened.

    Science.gov (United States)

    Visbal, Adriana P; LaMarca, Heather L; Villanueva, Hugo; Toneff, Michael J; Li, Yi; Rosen, Jeffrey M; Lewis, Michael T

    2011-04-01

    The Hedgehog (Hh) signaling network is critical for patterning and organogenesis in mammals, and has been implicated in a variety of cancers. Smoothened (Smo), the gene encoding the principal signal transducer, is overexpressed frequently in breast cancer, and constitutive activation in MMTV-SmoM2 transgenic mice caused alterations in mammary gland morphology, increased proliferation, and changes in stem/progenitor cell number. Both in transgenic mice and in clinical specimens, proliferative cells did not usually express detectable Smo, suggesting the hypothesis that Smo functioned in a non-cell autonomous manner to stimulate proliferation. Here, we employed a genetically tagged mouse model carrying a Cre-recombinase-dependent conditional allele of constitutively active Smo (SmoM2) to test this hypothesis. MMTV-Cre- or adenoviral-Cre-mediated SmoM2 expression in the luminal epithelium, but not in the myoepithelium, was required for the hyper-proliferative phenotypes. High levels of proliferation were observed in cells adjacent or in close-proximity to Smo expressing cells demonstrating that SmoM2 expressing cells were stimulating proliferation via a paracrine or juxtacrine mechanism. In contrast, Smo expression altered luminal cell differentiation in a cell-autonomous manner. SmoM2 expressing cells, purified by fluorescence activated cell sorting (FACS) via the genetic fluorescent tag, expressed high levels of Ptch2, Gli1, Gli2, Jag2 and Dll-1, and lower levels of Notch4 and Hes6, in comparison to wildtype cells. These studies provide insight into the mechanism of Smo activation in the mammary gland and its possible roles in breast tumorigenesis. In addition, these results also have potential implications for the interpretation of proliferative phenotypes commonly observed in other organs as a consequence of hedgehog signaling activation.

  3. An improved infrared technique for sorting pecans

    Science.gov (United States)

    Graeve, Thorsten; Dereniak, Eustace L.; Lamonica, John A., Jr.

    1991-10-01

    This paper presents the results of a study of pecan spectral reflectances. It describes an experiment for measuring the contrast between several components of raw pecan product to be sorted. An analysis of the experimental data reveals high contrast ratios in the infrared spectrum, suggesting a potential improvement in sorting efficiency when separating pecan meat from shells. It is believed that this technique has the potential to dramatically improve the efficiency of current sorting machinery, and to reduce the cost of processing pecans for the consumer market.

  4. Minimal Model Semantics for Sorted Constraint Representation

    Institute of Scientific and Technical Information of China (English)

    廖乐健; 史忠植

    1995-01-01

    Sorted constraint representation is a very useful representation in AI which combines class hierarchies and constraint networks.For such sorted constraint representation,a problem is how to generalize the idea of default inheritance to constraint network,where the attributes in a class or between different classes interact with each other via the network.To give a formal account for the defeasible reasoning in such representation,a general sorted constraint logic is proposed,and a minimal-model semantics for the logic is presented.

  5. Engineering a Cache-Oblivious Sorting Algorithm

    DEFF Research Database (Denmark)

    Brodal, Gerth Stølting; Fagerberg, Rolf; Vinther, Kristoffer

    2007-01-01

    This paper is an algorithmic engineering study of cache-oblivious sorting. We investigate by empirical methods a number of implementation issues and parameter choices for the cache-oblivious sorting algorithm Lazy Funnelsort, and compare the final algorithm with Quicksort, the established standard...... for comparison-based sorting, as well as with recent cache-aware proposals. The main result is a carefully implemented cache-oblivious sorting algorithm, which our experiments show can be faster than the best Quicksort implementation we are able to find, already for input sizes well within the limits of RAM....... It is also at least as fast as the recent cache-aware implementations included in the test. On disk the difference is even more pronounced regarding Quicksort and the cache-aware algorithms, whereas the algorithm is slower than a careful implementation of multiway Mergesort such as TPIE....

  6. Filter-less submicron hydrodynamic size sorting.

    Science.gov (United States)

    Fouet, M; Mader, M-A; Iraïn, S; Yanha, Z; Naillon, A; Cargou, S; Gué, A-M; Joseph, P

    2016-02-21

    We propose a simple microfluidic device able to separate submicron particles (critical size ∼0.1 μm) from a complex sample with no filter (minimum channel dimension being 5 μm) by hydrodynamic filtration. A model taking into account the actual velocity profile and hydrodynamic resistances enables prediction of the chip sorting properties for any geometry. Two design families are studied to obtain (i) small sizes within minutes (low-aspect ratio, two-level chip) and (ii) micron-sized sorting with a μL flow rate (3D architecture based on lamination). We obtain quantitative agreement of sorting performances both with experiments and with numerical solving, and determine the limits of the approach. We therefore demonstrate a passive, filter-less sub-micron size sorting with a simple, robust, and easy to fabricate design.

  7. Efficient sorting of Bessel beams [Conference paper

    CSIR Research Space (South Africa)

    Mhlanga, T

    2013-02-01

    Full Text Available A procedure to efficiently sort orbital angular momentum (OAM) states of light, by performing a Cartesian to log-polar coordinate transformation which translates helically phased beams into a transverse phase gradient, currently exists1. We...

  8. Quantum Database Search can do without Sorting

    CERN Document Server

    Patel, A

    2001-01-01

    Sorting is a fundamental computational process, which facilitates subsequent searching of a database. It can be thought of as factorisation of the search process. The location of a desired item in a sorted database can be found by classical queries that inspect one letter of the label at a time. For an unsorted database, no such classical quick search algorithm is available. If the database permits quantum queries, however, then mere digitisation is sufficient for efficient search. Sorting becomes redundant with the quantum superposition of states. A quantum algorithm is written down which locates the desired item in an unsorted database a factor of two faster than the best classical algorithm can in a sorted database. This algorithm has close resemblance to the assembly process in DNA replication.

  9. Syndecans: synergistic activators of cell adhesion

    DEFF Research Database (Denmark)

    Woods, A; Couchman, J R

    1998-01-01

    Cell-surface proteoglycans participate in cell adhesion, growth-factor signalling, lipase activity and anticoagulation. Until recently, only the roles of the glycosaminoglycan chains were investigated. Now, with molecular characterization of several core proteins, the roles of each individual...... molecules modulating integrin-based adhesion....

  10. Another Definition of Order—Sorted Algebra

    Institute of Scientific and Technical Information of China (English)

    何自强

    1998-01-01

    In this paper the definition of order-sorted algebra is generalized by introducing transformation functions between subtypes and supertypes.According to our definition,a type needn't be a subset of its supertype and a record model may form an order-sorted algebra.A new definition of equation is given.It has also been proved that equational theories and describing single inheritance have the initial model.

  11. Control of a brain-computer interface without spike sorting

    Science.gov (United States)

    Fraser, George W.; Chase, Steven M.; Whitford, Andrew; Schwartz, Andrew B.

    2009-10-01

    Two rhesus monkeys were trained to move a cursor using neural activity recorded with silicon arrays of 96 microelectrodes implanted in the primary motor cortex. We have developed a method to extract movement information from the recorded single and multi-unit activity in the absence of spike sorting. By setting a single threshold across all channels and fitting the resultant events with a spline tuning function, a control signal was extracted from this population using a Bayesian particle-filter extraction algorithm. The animals achieved high-quality control comparable to the performance of decoding schemes based on sorted spikes. Our results suggest that even the simplest signal processing is sufficient for high-quality neuroprosthetic control.

  12. Cancer cell exosomes depend on cell-surface heparan sulfate proteoglycans for their internalization and functional activity.

    Science.gov (United States)

    Christianson, Helena C; Svensson, Katrin J; van Kuppevelt, Toin H; Li, Jin-Ping; Belting, Mattias

    2013-10-22

    Extracellular vesicle (EV)-mediated intercellular transfer of signaling proteins and nucleic acids has recently been implicated in the development of cancer and other pathological conditions; however, the mechanism of EV uptake and how this may be targeted remain as important questions. Here, we provide evidence that heparan sulfate (HS) proteoglycans (PGs; HSPGs) function as internalizing receptors of cancer cell-derived EVs with exosome-like characteristics. Internalized exosomes colocalized with cell-surface HSPGs of the syndecan and glypican type, and exosome uptake was specifically inhibited by free HS chains, whereas closely related chondroitin sulfate had no effect. By using several cell mutants, we provide genetic evidence of a receptor function of HSPG in exosome uptake, which was dependent on intact HS, specifically on the 2-O and N-sulfation groups. Further, enzymatic depletion of cell-surface HSPG or pharmacological inhibition of endogenous PG biosynthesis by xyloside significantly attenuated exosome uptake. We provide biochemical evidence that HSPGs are sorted to and associate with exosomes; however, exosome-associated HSPGs appear to have no direct role in exosome internalization. On a functional level, exosome-induced ERK1/2 signaling activation was attenuated in PG-deficient mutant cells as well as in WT cells treated with xyloside. Importantly, exosome-mediated stimulation of cancer cell migration was significantly reduced in PG-deficient mutant cells, or by treatment of WT cells with heparin or xyloside. We conclude that cancer cell-derived exosomes use HSPGs for their internalization and functional activity, which significantly extends the emerging role of HSPGs as key receptors of macromolecular cargo.

  13. Automatic spike sorting using tuning information.

    Science.gov (United States)

    Ventura, Valérie

    2009-09-01

    Current spike sorting methods focus on clustering neurons' characteristic spike waveforms. The resulting spike-sorted data are typically used to estimate how covariates of interest modulate the firing rates of neurons. However, when these covariates do modulate the firing rates, they provide information about spikes' identities, which thus far have been ignored for the purpose of spike sorting. This letter describes a novel approach to spike sorting, which incorporates both waveform information and tuning information obtained from the modulation of firing rates. Because it efficiently uses all the available information, this spike sorter yields lower spike misclassification rates than traditional automatic spike sorters. This theoretical result is verified empirically on several examples. The proposed method does not require additional assumptions; only its implementation is different. It essentially consists of performing spike sorting and tuning estimation simultaneously rather than sequentially, as is currently done. We used an expectation-maximization maximum likelihood algorithm to implement the new spike sorter. We present the general form of this algorithm and provide a detailed implementable version under the assumptions that neurons are independent and spike according to Poisson processes. Finally, we uncover a systematic flaw of spike sorting based on waveform information only.

  14. Specified neural progenitors sort to form sharp domains after noisy Shh signaling.

    Science.gov (United States)

    Xiong, Fengzhu; Tentner, Andrea R; Huang, Peng; Gelas, Arnaud; Mosaliganti, Kishore R; Souhait, Lydie; Rannou, Nicolas; Swinburne, Ian A; Obholzer, Nikolaus D; Cowgill, Paul D; Schier, Alexander F; Megason, Sean G

    2013-04-25

    Sharply delineated domains of cell types arise in developing tissues under instruction of inductive signal (morphogen) gradients, which specify distinct cell fates at different signal levels. The translation of a morphogen gradient into discrete spatial domains relies on precise signal responses at stable cell positions. However, cells in developing tissues undergoing morphogenesis and proliferation often experience complex movements, which may affect their morphogen exposure, specification, and positioning. How is a clear pattern achieved with cells moving around? Using in toto imaging of the zebrafish neural tube, we analyzed specification patterns and movement trajectories of neural progenitors. We found that specified progenitors of different fates are spatially mixed following heterogeneous Sonic Hedgehog signaling responses. Cell sorting then rearranges them into sharply bordered domains. Ectopically induced motor neuron progenitors also robustly sort to correct locations. Our results reveal that cell sorting acts to correct imprecision of spatial patterning by noisy inductive signals.

  15. Active oxygen and cell death in cereal aleurone cells.

    Science.gov (United States)

    Fath, Angelika; Bethke, Paul; Beligni, Veronica; Jones, Russell

    2002-05-01

    The cereal aleurone layer is a secretory tissue whose function is regulated by gibberellic acid (GA) and abscisic acid (ABA). Aleurone cells lack functional chloroplasts, thus excluding photosynthesis as a source of active oxygen species (AOS) in cell death. Incubation of barley aleurone layers or protoplasts in GA initiated the cell death programme, but incubation in ABA delays programmed cell death (PCD). Light, especially blue and UV-A light, and H(2)O(2) accelerate PCD of GA-treated aleurone cells, but ABA-treated aleurone cells are refractory to light and H(2)O(2) and are not killed. It was shown that light elevated intracellular H(2)O(2), and that the rise in H(2)O(2) was greater in GA-treated cells compared to cells in ABA. Experiments with antioxidants show that PCD in aleurone is probably regulated by AOS. The sensitivity of GA-treated aleurone to light and H(2)O(2) is a result of lowered amounts of enzymes that metabolize AOS. mRNAs encoding catalase, ascorbate peroxidase and superoxide dismutase are all reduced during 6-18 h of incubation in GA, but these mRNAs were present in higher amounts in cells incubated in ABA. The amounts of protein and enzyme activities encoded by these mRNAs were also dramatically reduced in GA-treated cells. Aleurone cells store and metabolize neutral lipids via the glyoxylate cycle in response to GA, and glyoxysomes are one potential source of AOS in the GA-treated cells. Mitochondria are another potential source of AOS in GA-treated cells. AOS generated by these organelles bring about membrane rupture and cell death.

  16. Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes.

    Science.gov (United States)

    Stone, Matthew B; Shelby, Sarah A; Núñez, Marcos F; Wisser, Kathleen; Veatch, Sarah L

    2017-02-01

    Diverse cellular signaling events, including B cell receptor (BCR) activation, are hypothesized to be facilitated by domains enriched in specific plasma membrane lipids and proteins that resemble liquid-ordered phase-separated domains in model membranes. This concept remains controversial and lacks direct experimental support in intact cells. Here, we visualize ordered and disordered domains in mouse B lymphoma cell membranes using super-resolution fluorescence localization microscopy, demonstrate that clustered BCR resides within ordered phase-like domains capable of sorting key regulators of BCR activation, and present a minimal, predictive model where clustering receptors leads to their collective activation by stabilizing an extended ordered domain. These results provide evidence for the role of membrane domains in BCR signaling and a plausible mechanism of BCR activation via receptor clustering that could be generalized to other signaling pathways. Overall, these studies demonstrate that lipid mediated forces can bias biochemical networks in ways that broadly impact signal transduction.

  17. Mycoplasma suis infection results endothelial cell damage and activation: new insight into the cell tropism and pathogenicity of hemotrophic mycoplasma

    Directory of Open Access Journals (Sweden)

    Sokoli Albina

    2013-02-01

    Full Text Available Abstract Hemotrophic mycoplasmas (HM are highly specialized red blood cell parasites that cause infectious anemia in a variety of mammals, including humans. To date, no in vitro cultivation systems for HM have been available, resulting in relatively little information about the pathogenesis of HM infection. In pigs, Mycoplasma suis-induced infectious anemia is associated with hemorrhagic diathesis, and coagulation dysfunction. However, intravasal coagulation and subsequent consumption coagulopathy can only partly explain the sequence of events leading to hemorrhagic diathesis manifesting as cyanosis, petechial bleeding, and ecchymosis, and to disseminated coagulation. The involvement of endothelial activation and damage in M. suis-associated pathogenesis was investigated using light and electron microscopy, immunohistochemistry, and cell sorting. M. suis interacted directly with endothelial cells in vitro and in vivo. Endothelial activation, widespread endothelial damage, and adherence of red blood cells to the endothelium were evident in M. suis-infected pigs. These alterations of the endothelium were accompanied by hemorrhage, intravascular coagulation, vascular occlusion, and massive morphological changes within the parenchyma. M. suis biofilm-like microcolonies formed on the surface of endothelial cells, and may represent a putative persistence mechanism of M. suis. In vitro analysis demonstrated that M. suis interacted with the endothelial cytoskeletal protein actin, and induced actin condensation and activation of endothelial cells, as determined by the up-regulation of ICAM, PECAM, E-selectin, and P-selectin. These findings demonstrate an additional cell tropism of HM for endothelial cells and suggest that M. suis interferes with the protective function of the endothelium, resulting in hemorrhagic diathesis.

  18. Sorting and Manipulation of Magnetic Droplets in Continuous Flow

    Science.gov (United States)

    Al-Hetlani, Entesar; Hatt, Oliver J.; Vojtíšek, Martin; Tarn, Mark D.; Iles, Alexander; Pamme, Nicole

    2010-12-01

    We report the rapid on-chip generation and subsequent manipulation of magnetic droplets in continuous flow. Magnetic droplets were formed using aqueous-based ferrofluid as the dispersed phase and fluorocarbon oil as the continuous phase. Droplet manipulation was demonstrated with simple permanent magnets using two microfluidic platforms: (i) flow focusing droplet generation followed by their splitting into daughter droplets containing different amounts of magnetic nanoparticles, and (ii) droplet generation at a T-junction and their downstream deflection across a chamber for sorting based on the applied magnetic field and magnetite loading of the droplet. Both systems show great potential for performing a wide range of high throughput continuous flow processes including sample dilution, cell sorting and screening, and microparticle fabrication.

  19. Bursts of activity in collective cell migration

    CERN Document Server

    Chepizhko, Oleksandr; Mastrapasqua, Eleonora; Nourazar, Mehdi; Ascagni, Miriam; Sugni, Michela; Fascio, Umberto; Leggio, Livio; Malinverno, Chiara; Scita, Giorgio; Santucci, Stephane; Alava, Mikko J; Zapperi, Stefano; La Porta, Caterina A M

    2016-01-01

    Dense monolayers of living cells display intriguing relaxation dynamics, reminiscent of soft and glassy materials close to the jamming transition, and migrate collectively when space is available, as in wound healing or in cancer invasion. Here we show that collective cell migration occurs in bursts that are similar to those recorded in the propagation of cracks, fluid fronts in porous media and ferromagnetic domain walls. In analogy with these systems, the distribution of activity bursts displays scaling laws that are universal in different cell types and for cells moving on different substrates. The main features of the invasion dynamics are quantitatively captured by a model of interacting active particles moving in a disordered landscape. Our results illustrate that collective motion of living cells is analogous to the corresponding dynamics in driven, but inanimate, systems.

  20. Primary cortical brain cells influence osteoblast activity.

    Science.gov (United States)

    Anissian, Lucas; Kirby, Michael; Stark, André

    2009-12-18

    The presence of neuropeptides and neuroreceptors in the bone have been reported in several studies. Bone turn-over seems to be controlled by the nervous system. The actual pathway or the control mechanism is still under investigation. In this study we investigate the changes in osteoblast cells if they are in co-culture with primary cortical brain cells. After seven days in co-culture with the primary fetal brain cells the osteoblast cells exhibited hypertrophic morphological changes and showed stronger ALP activity.

  1. Dielectrophoresis microsystem with integrated flow cytometers for on-line monitoring of sorting efficiency

    DEFF Research Database (Denmark)

    Wang, Zhenyu; Hansen, Ole; Petersen, Peter Kalsen

    2006-01-01

    Dielectrophoresis (DEP) and flow cytometry are powerful technologies and widely applied in microfluidic systems for handling and measuring cells and particles. Here, we present a novel microchip with a DEP selective filter integrated with two microchip flow cytometers (FCs) for on-line monitoring...... of cell sorting processes. On the microchip, the DEP filter is integrated in a microfluidic channel network to sort yeast cells by positive DER The two FCs detection windows are set upstream and downstream of the DEP filter. When a cell passes through the detection windows, the light scattered by the cell...

  2. Aldehyde dehydrogenase activity selects for lung adenocarcinoma stem cells dependent on notch signaling.

    Science.gov (United States)

    Sullivan, James P; Spinola, Monica; Dodge, Michael; Raso, Maria G; Behrens, Carmen; Gao, Boning; Schuster, Katja; Shao, Chunli; Larsen, Jill E; Sullivan, Laura A; Honorio, Sofia; Xie, Yang; Scaglioni, Pier P; DiMaio, J Michael; Gazdar, Adi F; Shay, Jerry W; Wistuba, Ignacio I; Minna, John D

    2010-12-01

    Aldehyde dehydrogenase (ALDH) is a candidate marker for lung cancer cells with stem cell-like properties. Immunohistochemical staining of a large panel of primary non-small cell lung cancer (NSCLC) samples for ALDH1A1, ALDH3A1, and CD133 revealed a significant correlation between ALDH1A1 (but not ALDH3A1 or CD133) expression and poor prognosis in patients including those with stage I and N0 disease. Flow cytometric analysis of a panel of lung cancer cell lines and patient tumors revealed that most NSCLCs contain a subpopulation of cells with elevated ALDH activity, and that this activity is associated with ALDH1A1 expression. Isolated ALDH(+) lung cancer cells were observed to be highly tumorigenic and clonogenic as well as capable of self-renewal compared with their ALDH(-) counterparts. Expression analysis of sorted cells revealed elevated Notch pathway transcript expression in ALDH(+) cells. Suppression of the Notch pathway by treatment with either a γ-secretase inhibitor or stable expression of shRNA against NOTCH3 resulted in a significant decrease in ALDH(+) lung cancer cells, commensurate with a reduction in tumor cell proliferation and clonogenicity. Taken together, these findings indicate that ALDH selects for a subpopulation of self-renewing NSCLC stem-like cells with increased tumorigenic potential, that NSCLCs harboring tumor cells with ALDH1A1 expression have inferior prognosis, and that ALDH1A1 and CD133 identify different tumor subpopulations. Therapeutic targeting of the Notch pathway reduces this ALDH(+) component, implicating Notch signaling in lung cancer stem cell maintenance.

  3. Devices for the production and sorting of microfluidic droplets

    Science.gov (United States)

    Aubrecht, Donald; Heyman, John; Agresti, Jeremy; Köster, Sarah; Weitz, David

    2010-03-01

    Droplets produced in microfluidic devices are a great set of tools for studying large cell populations and permutations of reactions. Sample populations of 10^6 - 10^7 can be studied with relative ease, as encapsulation and screening rates in the kHz range are accessible. Previous droplet work has shown encapsulation of cells in droplets allows individual cells and their products to be studied. Advantages include correlation between detected products and initial drop contents, as well as minimized sample cross-contamination. Most microfluidic-based biological assays rely on fluorescent labeling of cells or use of cellular products to initiate a fluorescence-producing reaction. Detection of the fluorescence provides a trigger for sorting those cells or cell-containing droplets away from the general population. Though this allows some cellular processes to be studied, detection and quantification of all products, not just those expressed to the cell surface or those that catalyze reactions, would impact development of better therapeutics. We are currently working to adapt benchtop biological assays that label and detect cellular products for use in a droplet-based system. The work presented here details the chain of modular microfluidic devices we use to encapsulate, incubate, interrogate, and sort a population of droplets containing a model system.

  4. Interview: glycolipid antigen presentation by CD1d and the therapeutic potential of NKT cell activation.

    Science.gov (United States)

    Kronenberg, Mitchell

    2007-01-01

    Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious agents, and the development of artheriosclerotic plaques. In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens. Central to these studies is CD1d--the antigen presenting molecule that presents glycolipids to NKT cells. The advent of CD1d tetramer technology, a technique developed by the Kronenberg lab, is critical for the sorting and identification of subsets of specific glycolipid-reactive T cells. Mitch explains how glycolipid agonists are being used as therapeutic agents to activate NKT cells in cancer patients and how CD1d tetramers can be used to assess the state of the NKT cell population in vivo following glycolipid agonist therapy. Current status of ongoing clinical trials using these agonists are discussed as well as Mitch's prediction for areas in the field of immunology that will have emerging importance in the near future.

  5. Identification of Tumor Endothelial Cells with High Aldehyde Dehydrogenase Activity and a Highly Angiogenic Phenotype

    Science.gov (United States)

    Maishi, Nako; Ohga, Noritaka; Hida, Yasuhiro; Kawamoto, Taisuke; Iida, Junichiro; Shindoh, Masanobu; Tsuchiya, Kunihiko; Shinohara, Nobuo; Hida, Kyoko

    2014-01-01

    Tumor blood vessels play an important role in tumor progression and metastasis. It has been reported that tumor endothelial cells (TECs) exhibit highly angiogenic phenotypes compared with those of normal endothelial cells (NECs). TECs show higher proliferative and migratory abilities than those NECs, together with upregulation of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2). Furthermore, compared with NECs, stem cell markers such as Sca-1, CD90, and multidrug resistance 1 are upregulated in TECs, suggesting that stem-like cells exist in tumor blood vessels. In this study, to reveal the biological role of stem-like TECs, we analyzed expression of the stem cell marker aldehyde dehydrogenase (ALDH) in TECs and characterized ALDHhigh TECs. TECs and NECs were isolated from melanoma-xenografted nude mice and normal dermis, respectively. ALDH mRNA expression and activity were higher in TECs than those in NECs. Next, ALDHhigh/low TECs were isolated by fluorescence-activated cell sorting to compare their characteristics. Compared with ALDHlow TECs, ALDHhigh TECs formed more tubes on Matrigel-coated plates and sustained the tubular networks longer. Furthermore, VEGFR2 expression was higher in ALDHhigh TECs than that in ALDHlow TECs. In addition, ALDH was expressed in the tumor blood vessels of in vivo mouse models of melanoma and oral carcinoma, but not in normal blood vessels. These findings indicate that ALDHhigh TECs exhibit an angiogenic phenotype. Stem-like TECs may have an essential role in tumor angiogenesis. PMID:25437864

  6. Identification of tumor endothelial cells with high aldehyde dehydrogenase activity and a highly angiogenic phenotype.

    Directory of Open Access Journals (Sweden)

    Hitomi Ohmura-Kakutani

    Full Text Available Tumor blood vessels play an important role in tumor progression and metastasis. It has been reported that tumor endothelial cells (TECs exhibit highly angiogenic phenotypes compared with those of normal endothelial cells (NECs. TECs show higher proliferative and migratory abilities than those NECs, together with upregulation of vascular endothelial growth factor (VEGF and VEGF receptor 2 (VEGFR2. Furthermore, compared with NECs, stem cell markers such as Sca-1, CD90, and multidrug resistance 1 are upregulated in TECs, suggesting that stem-like cells exist in tumor blood vessels. In this study, to reveal the biological role of stem-like TECs, we analyzed expression of the stem cell marker aldehyde dehydrogenase (ALDH in TECs and characterized ALDHhigh TECs. TECs and NECs were isolated from melanoma-xenografted nude mice and normal dermis, respectively. ALDH mRNA expression and activity were higher in TECs than those in NECs. Next, ALDHhigh/low TECs were isolated by fluorescence-activated cell sorting to compare their characteristics. Compared with ALDHlow TECs, ALDHhigh TECs formed more tubes on Matrigel-coated plates and sustained the tubular networks longer. Furthermore, VEGFR2 expression was higher in ALDHhigh TECs than that in ALDHlow TECs. In addition, ALDH was expressed in the tumor blood vessels of in vivo mouse models of melanoma and oral carcinoma, but not in normal blood vessels. These findings indicate that ALDHhigh TECs exhibit an angiogenic phenotype. Stem-like TECs may have an essential role in tumor angiogenesis.

  7. 人胆管癌CD24+ CD44+ EpCAMhigh细胞亚群的分选及其肿瘤干细胞样特性的鉴定%Sorting of CD24+ CD44+ EpCAMhigh subset cells in human human cholangiocardnoma and the identificantion of their cancer stem cell-like properties

    Institute of Scientific and Technical Information of China (English)

    朱峰; 王敏; 秦仁义; 申铭; 王欣; 田锐; 江建新; 石程剑

    2010-01-01

    Objective To sort CD24+ CD44+ EpCAMhigh subset cells in human cholangiocarcinoma and identify their cancer stem cell-like properties. Methods The expression and rate of CD24, CD44 and EpCAM in 6 cases of human cholangiocacinomas were assayed by flow cytometry. The fresh specimens from two cases of cholangiocarcinoma were obtained and implanted subcutaneously into NOD/SCID mice for the establishment of xenografts model. CD24+ CD44+ EpCAMhigh subset cells were sorted from xenografts by flow cytometry and their tumorigenic potential, self-renewal ability and differentiation ability were assessed. Results The expression rate of CD24+ CD44+ EpCAMhigh cells ranged from 0. 58% to 2.43% (mean= 0. 94% ) in 6 cholangiocarcinoma specimens and 2 xenografts. CD24+ CD44+ EpCAMhigh subset cells sorted from 2 xenografts were found to be highly tumorigenic in NOD/SCID mice. CD24+ CD44+ EpCAMhigh cells consistently formed tumors with 1000 cells in 3/8 mice. In contrast, CD24+ CD44+ EpCAMhigh tumor cells were less tumorigenic and formed tumors with 50 000 cells in 1/8 mice. CD24+ CD44+ EpCAMhigh cells were passaged in NOD/SCID mice and formed tumors that recapitulated the histological features and heterogeneity of the original patient tumor. Conclusion CD24+ CD44+ EpCAMhigh subset cells were discriminated in human cholangiocarcinoma, and they had highly tumorigenic, self-renewal ability and differentiation ability. It was first confirmed that CD24+ CD44+ EpCAMhigh cells may be human cholangiocarcinoma cancer stem cells.%目的 检测及分选人胆管癌中的CD24+ CD44+ EpCAMhigh细胞亚群,探讨其是否具有肿瘤干细胞样生物学特性.方法 流式细胞术检测6例人胆管癌中CD24、CD44、EpCAM的表达率;取2例人胆管癌新鲜标本种植到NOD/SCID鼠皮下,建立荷人胆管癌小鼠模型.流式细胞术分选CD24+ CD44+ EpCAMhigh亚群细胞,NOD/SCID鼠移植瘤试验鉴定其成瘤和分化能力.结果 6例人胆管癌组织标本和2例移植瘤中,CD24

  8. Upregulation of immunoproteasome subunits in myositis indicates active inflammation with involvement of antigen presenting cells, CD8 T-cells and IFNΓ.

    Directory of Open Access Journals (Sweden)

    Khetam Ghannam

    Full Text Available OBJECTIVE: In idiopathic inflammatory myopathies (IIM infiltration of immune cells into muscle and upregulation of MHC-I expression implies increased antigen presentation and involvement of the proteasome system. To decipher the role of immunoproteasomes in myositis, we investigated individual cell types and muscle tissues and focused on possible immune triggers. METHODS: Expression of constitutive (PSMB5, -6, -7 and corresponding immunoproteasomal subunits (PSMB8, -9, -10 was analyzed by real-time RT-PCR in muscle biopsies and sorted peripheral blood cells of patients with IIM, non-inflammatory myopathies (NIM and healthy donors (HD. Protein analysis in muscle biopsies was performed by western blot. Affymetrix HG-U133 platform derived transcriptome data from biopsies of different muscle diseases and from immune cell types as well as monocyte stimulation experiments were used for validation, coregulation and coexpression analyses. RESULTS: Real-time RT-PCR revealed significantly increased expression of immunoproteasomal subunits (PSMB8/-9/-10 in DC, monocytes and CD8+ T-cells in IIM. In muscle biopsies, the immunosubunits were elevated in IIM compared to NIM and exceeded levels of matched blood samples. Proteins of PSMB8 and -9 were found only in IIM but not NIM muscle biopsies. Reanalysis of 78 myositis and 20 healthy muscle transcriptomes confirmed these results and revealed involvement of the antigen processing and presentation pathway. Comparison with reference profiles of sorted immune cells and healthy muscle confirmed upregulation of PSMB8 and -9 in myositis biopsies beyond infiltration related changes. This upregulation correlated highest with STAT1, IRF1 and IFNγ expression. Elevation of T-cell specific transcripts in active IIM muscles was accompanied by increased expression of DC and monocyte marker genes and thus reflects the cell type specific involvement observed in peripheral blood. CONCLUSIONS: Immunoproteasomes seem to indicate

  9. The Role of the Clathrin Adaptor AP-1: Polarized Sorting and Beyond

    Directory of Open Access Journals (Sweden)

    Fubito Nakatsu

    2014-11-01

    Full Text Available The selective transport of proteins or lipids by vesicular transport is a fundamental process supporting cellular physiology. The budding process involves cargo sorting and vesicle formation at the donor membrane and constitutes an important process in vesicular transport. This process is particularly important for the polarized sorting in epithelial cells, in which the cargo molecules need to be selectively sorted and transported to two distinct destinations, the apical or basolateral plasma membrane. Adaptor protein (AP-1, a member of the AP complex family, which includes the ubiquitously expressed AP-1A and the epithelium-specific AP-1B, regulates polarized sorting at the trans-Golgi network and/or at the recycling endosomes. A growing body of evidence, especially from studies using model organisms and animals, demonstrates that the AP-1-mediated polarized sorting supports the development and physiology of multi-cellular units as functional organs and tissues (e.g., cell fate determination, inflammation and gut immune homeostasis. Furthermore, a possible involvement of AP-1B in the pathogenesis of human diseases, such as Crohn’s disease and cancer, is now becoming evident. These data highlight the significant contribution of AP-1 complexes to the physiology of multicellular organisms, as master regulators of polarized sorting in epithelial cells.

  10. Identification of residual leukemic cells by flow cytometry in childhood B-cell precursor acute lymphoblastic leukemia: verification of leukemic state by flow-sorting and molecular/cytogenetic methods

    OpenAIRE

    2012-01-01

    Reduction in minimal residual disease, measured by real-time quantitative PCR or flow cytometry, predicts prognosis in childhood B-cell precursor acute lymphoblastic leukemia. We explored whether cells reported as minimal residual disease by flow cytometry represent the malignant clone harboring clone-specific genomic markers (53 follow-up bone marrow samples from 28 children with B-cell precursor acute lymphoblastic leukemia). Cell populations (presumed leukemic and non-leukemic) were flow-s...

  11. T cell activation in APECED patients

    OpenAIRE

    Mannerström, Helga

    2013-01-01

    Autoimmune polyendocrinopathy-candidasis-ectodermal dystrophy, APECED, is a rare monogenic autoimmune disease in humans, which is caused by loss-of-function mutation in Autoimmune Regulator gene, AIRE. Previous results have shown impairments in the circulating T cells of the APECED patients. In this study we wanted to look closer on the disturbance in the T cell receptor development of APECED patients. By studying the TCR-mediated responsiveness of CD3 stimulation and comparing the activation...

  12. Entangled active matter: From cells to ants

    Science.gov (United States)

    Hu, D. L.; Phonekeo, S.; Altshuler, E.; Brochard-Wyart, F.

    2016-07-01

    Both cells and ants belong to the broad field of active matter, a novel class of non-equilibrium materials composed of many interacting units that individually consume energy and collectively generate motion or mechanical stresses. However cells and ants differ from fish and birds in that they can support static loads. This is because cells and ants can be entangled, so that individual units are bound by transient links. Entanglement gives cells and ants a set of remarkable properties usually not found together, such as the ability to flow like a fluid, spring back like an elastic solid, and self-heal. In this review, we present the biology, mechanics and dynamics of both entangled cells and ants. We apply concepts from soft matter physics and wetting to characterize these systems as well as to point out their differences, which arise from their differences in size. We hope that our viewpoints will spur further investigations into cells and ants as active materials, and inspire the fabrication of synthetic active matter.

  13. Critical telomerase activity for uncontrolled cell growth

    Science.gov (United States)

    Wesch, Neil L.; Burlock, Laura J.; Gooding, Robert J.

    2016-08-01

    The lengths of the telomere regions of chromosomes in a population of cells are modelled using a chemical master equation formalism, from which the evolution of the average number of cells of each telomere length is extracted. In particular, the role of the telomere-elongating enzyme telomerase on these dynamics is investigated. We show that for biologically relevant rates of cell birth and death, one finds a critical rate, R crit, of telomerase activity such that the total number of cells diverges. Further, R crit is similar in magnitude to the rates of mitosis and cell death. The possible relationship of this result to replicative immortality and its associated hallmark of cancer is discussed.

  14. A Success of Some Sort

    NARCIS (Netherlands)

    Venot, J.P.

    2016-01-01

    This paper explains the processes behind the framing of drip irrigation as a promising technology to address current poverty and environmental challenges in the developing world. I draw from critical development and science and technology studies and highlight that this imagery has been actively

  15. A Success of Some Sort

    NARCIS (Netherlands)

    Venot, J.P.

    2016-01-01

    This paper explains the processes behind the framing of drip irrigation as a promising technology to address current poverty and environmental challenges in the developing world. I draw from critical development and science and technology studies and highlight that this imagery has been actively

  16. Sorting Potatoes for Miss Bonner.

    Science.gov (United States)

    Herreid, Clyde Freeman

    1998-01-01

    Discusses the basis of a classification scheme for types of case studies. Four major classification headings are identified: (1) individual assignment; (2) lecture; (3) discussion; and (4) small group activities. Describes each heading from the point of view of several teaching methods. (DDR)

  17. Intracellular mechanisms of lymphoid cell activation.

    Science.gov (United States)

    Fresa, K; Hameed, M; Cohen, S

    1989-01-01

    Activation of lymphocytes for proliferation is associated with the appearance of an intracellular factor (ADR) that can induce DNA synthesis in isolated quiescent nuclei. ADR plays a role in the sequence of intracellular events leading to activation for IL-2-mediated proliferation. Because of the nature of the defining assay, the locus of ADR action appears to be near the terminal end of the transduction pathway. Interestingly, although lymphocytes from aged individuals respond poorly to proliferative stimuli, they appear to produce normal to above-normal levels of ADR. In contrast, their nuclei are only poorly responsive to stimulation by ADR. Preparations rich in ADR activity have proteolytic activity as well. In addition, aprotinin, as well as a variety of other protease inhibitors, suppresses ADR-induced DNA synthesis in a dose-dependent manner. ADR activity can be removed from active extracts by absorption with aprotinin-conjugated agarose beads, and can be removed from the beads by elution at pH 5.0. This latter suggests that ADR itself is a protease. However, its endogenous substrate is not yet known. We have also detected an inhibitor of ADR activity in the cytoplasm of resting lymphocytes. This is a heat-stable protein of approximately 60,000 Da. In addition to suppressing the interaction of ADR with quiescent nuclei, the inhibitor can suppress DNA synthetic activity of replicative nuclei isolated from mitogen-activated lymphocytes. Interestingly, these preparations had little or no activity on replicative nuclei derived from several neoplastic cell lines. The resistance of tumor cell nuclei to spontaneously occurring cytoplasmic inhibitory factors such as the one described here may provide one explanation for the loss of growth control in neoplastic cells.

  18. Order-Sorted Parameterization and Induction

    Science.gov (United States)

    Meseguer, José

    Parameterization is one of the most powerful features to make specifications and declarative programs modular and reusable, and our best hope for scaling up formal verification efforts. This paper studies order-sorted parameterization at three different levels: (i) its mathematical semantics; (ii) its operational semantics by term rewriting; and (iii) the inductive reasoning principles that can soundly be used to prove properties about such specifications. It shows that achieving the desired properties at each of these three levels is a considerably subtler matter than for many-sorted specifications, but that such properties can be attained under reasonable conditions.

  19. MODELING WORK OF SORTING STATION USING UML

    Directory of Open Access Journals (Sweden)

    O. V. Gorbova

    2014-12-01

    Full Text Available Purpose. The purpose of this paper is the construction of methods and models for the graphical representation process of sorting station, using the unified modeling language (UML. Methodology. Methods of graph theory, finite automata and the representation theory of queuing systems were used as the methods of investigation. A graphical representation of the process was implemented with using the Unified Modeling Language UML. The sorting station process representation is implemented as a state diagram and actions through a set of IBM Rational Rose. Graphs can show parallel operation of sorting station, the parallel existence and influence of objects process and the transition from one state to another. The IBM Rational Rose complex allows developing a diagram of work sequence of varying degrees of detailing. Findings. The study has developed a graphical representation method of the process of sorting station of different kind of complexity. All graphical representations are made using the UML. They are represented as a directed graph with the states. It is clear enough in the study of the subject area. Applying the methodology of the representation process, it allows becoming friendly with the work of any automation object very fast, and exploring the process during algorithms construction of sorting stations and other railway facilities. This model is implemented with using the Unified Modeling Language (UML using a combination of IBM Rational Rose. Originality. The representation process of sorting station was developed by means of the Unified Modeling Language (UML use. Methodology of representation process allows creating the directed graphs based on the order of execution of the works chain, objects and performers of these works. The UML allows visualizing, specifying, constructing and documenting, formalizing the representation process of sorting station and developing sequence diagrams of works of varying degrees of detail. Practical