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    Becton Dickinson facsaria
    IL-6Rα high effector memory (EM) CD8 + T cells selectively produce high levels of IL-5 and IL-13 as well as potently proliferate and survive in response to T-cell receptor triggering. ( A–E ) Peripheral blood mononuclear cells were obtained from healthy donors. ( A ) Multiplex cytokine assay of culture supernatants from fluorescence-activated cell sorter (FACS) purified naive, IL-6Rα high , IL-6Rα low IL-7Rα high , and IL-6Rα low IL-7Rα low EM CD8 + T cells that were stimulated for 5 days with anti-CD3/CD28 antibody (Ab)-coated beads or phosphate-buffered saline (unstimulated). ( B ) Frequency of proliferating cells in FACS-purified naive, IL-6Rα high , IL-6Rα low IL-7Rα high , and IL-6Rα low IL-7Rα low EM CD8 + T cells that were stained with CFSE and stimulated for 5 days with or without anti-CD3/CD28 Ab-coated beads. The frequency of proliferating cells was determined by flow cytometry. ( C ) IL-2 measurement of culture supernatants from cells stimulated as in A . ( D ) Flow cytometric analysis of proliferating cells in FACS-purified IL-6Rα high , IL-6Rα low IL-7Rα high , and IL-6Rα low IL-7Rα low EM CD8 + T cells that were stained with CFSE and stimulated for 5 days with or without anti-CD3/CD28 Ab-coated beads in the presence or absence of anti–IL-2 Abs. ( E ) Frequency of live cells (Annexin V − and 7AAD − ) in FACS-purified IL-6Rα high , IL-6Rα low IL-7Rα high , and IL-6Rα low IL-7Rα low EM CD8 + T cells that were stimulated for 5 days with or without anti-CD3/CD28 Ab-coated beads. The frequency of live cells was determined by staining with Annexin V and 7AAD. FACS sorting and flow cytometry were done using <t>FACSAria</t> and LSRII, respectively. Numbers in dot plots indicate the frequency of live cells. Bars and error bars indicate mean (n = 5–8 donors) ± SEM ( A–C ). Representative data from two independent experiments with two donors ( D and E ). *Undetectable levels of cytokines. P values were obtained by one-way analysis of variance. ** P
    Facsaria, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Becton Dickinson facsaria ii
    Image-based capture of individual spiked cells . (A) Cell surface GD2 expression was measured by flow cytometry for the eight neuroblastoma cell lines used for isolation of single cells (and listed at right), as compared to negative control WBCs and isotype control. (B) WBCs and the GD2-dim neuroblastoma cell line SY5Y were mixed at a tumor:WBC ratio of 1:35, pre-labeled with GD2-PE, CD45-FITC, and Hoechst nuclear dye, then injected into the DEPArray. Shown are bright-field images of (i) a single tumor cell (bar depicts 10 μm) and (ii) a cluster of two cells. In (C) , a scatter plot of GD2-PE and CD45-FITC mean fluorescence intensity (MFI) is shown as well as cell images, including on the left-hand side of the dot-plot (i) an image of a single tumor cell and (ii) a single WBC, and on the right-hand side (iii) a heterogeneous cluster, (iv) a homogeneous cluster, and (v) a spurious event. (D) NB1643M cells and normal donor WBCs were mixed at a ratio of 1:1,000,000 and stained with GD2-PE and CD45-FITC, in this representative experiment. The sample was pre-enriched using <t>FACSAria-based</t> sorting, and the enriched fraction placed into the DEPArray cartridge. Shown are a scatter plot of GD2-PE and CD45-FITC MFI, as well as images of (i–iv) intact tumor cells, (v and vi) WBCs, and (vii–ix) debris and false-positive events.
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    86
    Becton Dickinson facsaria cell sorter
    Behaviour of an inverted ON switch generated via insertion of the module into an OFF switch. ( a ) Construction of the INPUT and OUTPUT plasmids transfected into HeLa cells in this study. The INPUT plasmids express either L7Ae or the MS2 coat protein (MS2) as a cognate or noncognate RNA-binding protein, respectively. DsRed-Express is also synthesized from the INPUT plasmid, driven by the IRES. An OUTPUT plasmid expresses ON-switch mRNAs containing K-turn (Kt) or defective K-turn (dKt) as either an active or defective sensory motif, respectively. EGFP driven by the IRES is synthesized from the OUTPUT plasmid. ( b – e ) Images of cells captured via fluorescence microscopy. DsRed-Express synthesized together with L7Ae ( c , e ) or MS2 ( b , d ) is shown in red. EGFP outputs from an ON switch with either an active ( b , c ; ON-Kt) or a defective ( d , e ; ON-dKt) sensor are shown in green. These two fluorescent signals are merged and shown in the right most pictures. Scale bars, 200 μm. ( f ) The mean intensity of EGFP fluorescence of transfected cells. HeLa cells shown in b – e were analysed using a flow cytometer, <t>FACSAria.</t> Cells expressing only DsRed-Express were used as the negative control (DsRed-Express). Bars and error bars represent the mean and s.d., respectively, of three triplicate experiments ( n =9). ( g ) The levels of ON-switch mRNAs, as determined by quantitative RT–PCR. To eliminate the noise from untransfected cells, the results are normalized by employing the mRNA of neomycin-resistant gene transcribed from the OUTPUT plasmid in cells. Bars and error bars represent the mean and s.d., respectively, of three triplicate experiments ( n =9). n.a., not analysed. All experiments in b – g were carried out 24 h after the transfection of 100 ng of the indicated OUTPUT plasmid and 20 ng of the INPUT plasmid together with 480 ng of the noncognate INPUT plasmid.
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    Becton Dickinson flow cytometry
    Behaviour of an inverted ON switch generated via insertion of the module into an OFF switch. ( a ) Construction of the INPUT and OUTPUT plasmids transfected into HeLa cells in this study. The INPUT plasmids express either L7Ae or the MS2 coat protein (MS2) as a cognate or noncognate RNA-binding protein, respectively. DsRed-Express is also synthesized from the INPUT plasmid, driven by the IRES. An OUTPUT plasmid expresses ON-switch mRNAs containing K-turn (Kt) or defective K-turn (dKt) as either an active or defective sensory motif, respectively. EGFP driven by the IRES is synthesized from the OUTPUT plasmid. ( b – e ) Images of cells captured via fluorescence microscopy. DsRed-Express synthesized together with L7Ae ( c , e ) or MS2 ( b , d ) is shown in red. EGFP outputs from an ON switch with either an active ( b , c ; ON-Kt) or a defective ( d , e ; ON-dKt) sensor are shown in green. These two fluorescent signals are merged and shown in the right most pictures. Scale bars, 200 μm. ( f ) The mean intensity of EGFP fluorescence of transfected cells. HeLa cells shown in b – e were analysed using a flow cytometer, <t>FACSAria.</t> Cells expressing only DsRed-Express were used as the negative control (DsRed-Express). Bars and error bars represent the mean and s.d., respectively, of three triplicate experiments ( n =9). ( g ) The levels of ON-switch mRNAs, as determined by quantitative RT–PCR. To eliminate the noise from untransfected cells, the results are normalized by employing the mRNA of neomycin-resistant gene transcribed from the OUTPUT plasmid in cells. Bars and error bars represent the mean and s.d., respectively, of three triplicate experiments ( n =9). n.a., not analysed. All experiments in b – g were carried out 24 h after the transfection of 100 ng of the indicated OUTPUT plasmid and 20 ng of the INPUT plasmid together with 480 ng of the noncognate INPUT plasmid.
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    IL-6Rα high effector memory (EM) CD8 + T cells selectively produce high levels of IL-5 and IL-13 as well as potently proliferate and survive in response to T-cell receptor triggering. ( A–E ) Peripheral blood mononuclear cells were obtained from healthy donors. ( A ) Multiplex cytokine assay of culture supernatants from fluorescence-activated cell sorter (FACS) purified naive, IL-6Rα high , IL-6Rα low IL-7Rα high , and IL-6Rα low IL-7Rα low EM CD8 + T cells that were stimulated for 5 days with anti-CD3/CD28 antibody (Ab)-coated beads or phosphate-buffered saline (unstimulated). ( B ) Frequency of proliferating cells in FACS-purified naive, IL-6Rα high , IL-6Rα low IL-7Rα high , and IL-6Rα low IL-7Rα low EM CD8 + T cells that were stained with CFSE and stimulated for 5 days with or without anti-CD3/CD28 Ab-coated beads. The frequency of proliferating cells was determined by flow cytometry. ( C ) IL-2 measurement of culture supernatants from cells stimulated as in A . ( D ) Flow cytometric analysis of proliferating cells in FACS-purified IL-6Rα high , IL-6Rα low IL-7Rα high , and IL-6Rα low IL-7Rα low EM CD8 + T cells that were stained with CFSE and stimulated for 5 days with or without anti-CD3/CD28 Ab-coated beads in the presence or absence of anti–IL-2 Abs. ( E ) Frequency of live cells (Annexin V − and 7AAD − ) in FACS-purified IL-6Rα high , IL-6Rα low IL-7Rα high , and IL-6Rα low IL-7Rα low EM CD8 + T cells that were stimulated for 5 days with or without anti-CD3/CD28 Ab-coated beads. The frequency of live cells was determined by staining with Annexin V and 7AAD. FACS sorting and flow cytometry were done using FACSAria and LSRII, respectively. Numbers in dot plots indicate the frequency of live cells. Bars and error bars indicate mean (n = 5–8 donors) ± SEM ( A–C ). Representative data from two independent experiments with two donors ( D and E ). *Undetectable levels of cytokines. P values were obtained by one-way analysis of variance. ** P

    Journal: American Journal of Respiratory and Critical Care Medicine

    Article Title: IL-6 Receptor α Defines Effector Memory CD8+ T Cells Producing Th2 Cytokines and Expanding in Asthma

    doi: 10.1164/rccm.201403-0601OC

    Figure Lengend Snippet: IL-6Rα high effector memory (EM) CD8 + T cells selectively produce high levels of IL-5 and IL-13 as well as potently proliferate and survive in response to T-cell receptor triggering. ( A–E ) Peripheral blood mononuclear cells were obtained from healthy donors. ( A ) Multiplex cytokine assay of culture supernatants from fluorescence-activated cell sorter (FACS) purified naive, IL-6Rα high , IL-6Rα low IL-7Rα high , and IL-6Rα low IL-7Rα low EM CD8 + T cells that were stimulated for 5 days with anti-CD3/CD28 antibody (Ab)-coated beads or phosphate-buffered saline (unstimulated). ( B ) Frequency of proliferating cells in FACS-purified naive, IL-6Rα high , IL-6Rα low IL-7Rα high , and IL-6Rα low IL-7Rα low EM CD8 + T cells that were stained with CFSE and stimulated for 5 days with or without anti-CD3/CD28 Ab-coated beads. The frequency of proliferating cells was determined by flow cytometry. ( C ) IL-2 measurement of culture supernatants from cells stimulated as in A . ( D ) Flow cytometric analysis of proliferating cells in FACS-purified IL-6Rα high , IL-6Rα low IL-7Rα high , and IL-6Rα low IL-7Rα low EM CD8 + T cells that were stained with CFSE and stimulated for 5 days with or without anti-CD3/CD28 Ab-coated beads in the presence or absence of anti–IL-2 Abs. ( E ) Frequency of live cells (Annexin V − and 7AAD − ) in FACS-purified IL-6Rα high , IL-6Rα low IL-7Rα high , and IL-6Rα low IL-7Rα low EM CD8 + T cells that were stimulated for 5 days with or without anti-CD3/CD28 Ab-coated beads. The frequency of live cells was determined by staining with Annexin V and 7AAD. FACS sorting and flow cytometry were done using FACSAria and LSRII, respectively. Numbers in dot plots indicate the frequency of live cells. Bars and error bars indicate mean (n = 5–8 donors) ± SEM ( A–C ). Representative data from two independent experiments with two donors ( D and E ). *Undetectable levels of cytokines. P values were obtained by one-way analysis of variance. ** P

    Article Snippet: EM CD8+ T-cell subsets were sorted using a FACSAria (BD Biosciences, San Jose, CA) ( see Figure E2 in the online supplement for gating strategy).

    Techniques: Multiplex Assay, Cytokine Assay, Fluorescence, FACS, Purification, Staining, Flow Cytometry, Cytometry

    Identification of effector memory (EM) CD8 + T cells with high levels of IL-6 receptor α (IL-6Rα) expression in healthy human peripheral blood. ( A ) Flow cytometric analysis of IL-6Rα and IL-7Rα expression on EM (CD45RA +/− CCR7 − ) CD8 + T cells in peripheral blood of a healthy donor. ( B ) Frequency of IL-6Rα high , IL-6Rα low IL-7Rα high , and IL-6Rα low IL-7Rα low cells in EM CD8 + T cells of healthy donors (n = 12) as measured by flow cytometry. ( C ) Western blot analysis of IL-6Rα expression by fluorescence-activated cell sorter (FACS) sorted IL-6Rα high , IL-6Rα low IL-7Rα high , and IL-6Rα low IL-7Rα low EM CD8 + T cells. ( D ) IL6RA gene expression in FACS-sorted IL-6Rα high and IL-6Rα low IL-7Rα high EM CD8 + T cells from healthy donors (n = 9) as measured by quantitative polymerase chain reaction. ( E ) Flow cytometric analysis of CD45RA on IL-6Rα high and IL-6Rα low IL-7Rα high EM CD8 + T cells. ( F ) Flow cytometric analysis of gp130 expression on IL-6Rα high , IL-6Rα low IL-7Rα high , and IL-6Rα low IL-7Rα low cells in EM CD8 + T cells. FACS sorting and flow cytometry were done using FACSAria and LSRII, respectively. Representative data from more than 10 ( A , E , F ) or 4 ( C ) independent experiments. Bars and error bars indicate mean ± SEM. P value was obtained by Student t test.

    Journal: American Journal of Respiratory and Critical Care Medicine

    Article Title: IL-6 Receptor α Defines Effector Memory CD8+ T Cells Producing Th2 Cytokines and Expanding in Asthma

    doi: 10.1164/rccm.201403-0601OC

    Figure Lengend Snippet: Identification of effector memory (EM) CD8 + T cells with high levels of IL-6 receptor α (IL-6Rα) expression in healthy human peripheral blood. ( A ) Flow cytometric analysis of IL-6Rα and IL-7Rα expression on EM (CD45RA +/− CCR7 − ) CD8 + T cells in peripheral blood of a healthy donor. ( B ) Frequency of IL-6Rα high , IL-6Rα low IL-7Rα high , and IL-6Rα low IL-7Rα low cells in EM CD8 + T cells of healthy donors (n = 12) as measured by flow cytometry. ( C ) Western blot analysis of IL-6Rα expression by fluorescence-activated cell sorter (FACS) sorted IL-6Rα high , IL-6Rα low IL-7Rα high , and IL-6Rα low IL-7Rα low EM CD8 + T cells. ( D ) IL6RA gene expression in FACS-sorted IL-6Rα high and IL-6Rα low IL-7Rα high EM CD8 + T cells from healthy donors (n = 9) as measured by quantitative polymerase chain reaction. ( E ) Flow cytometric analysis of CD45RA on IL-6Rα high and IL-6Rα low IL-7Rα high EM CD8 + T cells. ( F ) Flow cytometric analysis of gp130 expression on IL-6Rα high , IL-6Rα low IL-7Rα high , and IL-6Rα low IL-7Rα low cells in EM CD8 + T cells. FACS sorting and flow cytometry were done using FACSAria and LSRII, respectively. Representative data from more than 10 ( A , E , F ) or 4 ( C ) independent experiments. Bars and error bars indicate mean ± SEM. P value was obtained by Student t test.

    Article Snippet: EM CD8+ T-cell subsets were sorted using a FACSAria (BD Biosciences, San Jose, CA) ( see Figure E2 in the online supplement for gating strategy).

    Techniques: Expressing, Flow Cytometry, Cytometry, Western Blot, Fluorescence, FACS, Real-time Polymerase Chain Reaction

    A combination of IL-6 and IL-15 potently induces proliferation of IL-6Rα high EM CD8 + T cells that produce high levels of IL-5 and IL-13 in response to respiratory syncytial virus (RSV) and bacterial superantigens. ( A ) The frequency of proliferating cells in fluorescence-activated cell sorter (FACS) purified IL-6Rα high EM CD8 + T cells that were cultured for 7 days in the presence or absence of IL-6 (25 ng/ml) and/or IL-15 (5 ng/ml) or with anti-CD3/CD28 antibody-coated beads. ( B , C , and D ) Multiplex cytokine assay of culture supernatants from FACS-purified IL-6Rα high , IL-6Rα low IL-7Rα high , and IL-6Rα low IL-7Rα low EM CD8 + T cells that were incubated for 7 days with RSV lysate ( B ), staphylococcal enterotoxin B (SEB) ( C ), toxic shock syndrome toxin-1 (TSST-1) ( D ), or phosphate-buffered saline (control) in the presence of mitomycin C–treated autologous peripheral blood mononuclear cells as antigen-presenting cells (APCs). FACS sorting was done using FACSAria. Representative data from two ( A ) and four ( B , C , and D ) independent experiments with two and four healthy donors, respectively. *Undetectable levels of cytokines.

    Journal: American Journal of Respiratory and Critical Care Medicine

    Article Title: IL-6 Receptor α Defines Effector Memory CD8+ T Cells Producing Th2 Cytokines and Expanding in Asthma

    doi: 10.1164/rccm.201403-0601OC

    Figure Lengend Snippet: A combination of IL-6 and IL-15 potently induces proliferation of IL-6Rα high EM CD8 + T cells that produce high levels of IL-5 and IL-13 in response to respiratory syncytial virus (RSV) and bacterial superantigens. ( A ) The frequency of proliferating cells in fluorescence-activated cell sorter (FACS) purified IL-6Rα high EM CD8 + T cells that were cultured for 7 days in the presence or absence of IL-6 (25 ng/ml) and/or IL-15 (5 ng/ml) or with anti-CD3/CD28 antibody-coated beads. ( B , C , and D ) Multiplex cytokine assay of culture supernatants from FACS-purified IL-6Rα high , IL-6Rα low IL-7Rα high , and IL-6Rα low IL-7Rα low EM CD8 + T cells that were incubated for 7 days with RSV lysate ( B ), staphylococcal enterotoxin B (SEB) ( C ), toxic shock syndrome toxin-1 (TSST-1) ( D ), or phosphate-buffered saline (control) in the presence of mitomycin C–treated autologous peripheral blood mononuclear cells as antigen-presenting cells (APCs). FACS sorting was done using FACSAria. Representative data from two ( A ) and four ( B , C , and D ) independent experiments with two and four healthy donors, respectively. *Undetectable levels of cytokines.

    Article Snippet: EM CD8+ T-cell subsets were sorted using a FACSAria (BD Biosciences, San Jose, CA) ( see Figure E2 in the online supplement for gating strategy).

    Techniques: Fluorescence, FACS, Purification, Cell Culture, Multiplex Assay, Cytokine Assay, Incubation

    Image-based capture of individual spiked cells . (A) Cell surface GD2 expression was measured by flow cytometry for the eight neuroblastoma cell lines used for isolation of single cells (and listed at right), as compared to negative control WBCs and isotype control. (B) WBCs and the GD2-dim neuroblastoma cell line SY5Y were mixed at a tumor:WBC ratio of 1:35, pre-labeled with GD2-PE, CD45-FITC, and Hoechst nuclear dye, then injected into the DEPArray. Shown are bright-field images of (i) a single tumor cell (bar depicts 10 μm) and (ii) a cluster of two cells. In (C) , a scatter plot of GD2-PE and CD45-FITC mean fluorescence intensity (MFI) is shown as well as cell images, including on the left-hand side of the dot-plot (i) an image of a single tumor cell and (ii) a single WBC, and on the right-hand side (iii) a heterogeneous cluster, (iv) a homogeneous cluster, and (v) a spurious event. (D) NB1643M cells and normal donor WBCs were mixed at a ratio of 1:1,000,000 and stained with GD2-PE and CD45-FITC, in this representative experiment. The sample was pre-enriched using FACSAria-based sorting, and the enriched fraction placed into the DEPArray cartridge. Shown are a scatter plot of GD2-PE and CD45-FITC MFI, as well as images of (i–iv) intact tumor cells, (v and vi) WBCs, and (vii–ix) debris and false-positive events.

    Journal: Frontiers in Oncology

    Article Title: Dielectrophoretic Capture and Genetic Analysis of Single Neuroblastoma Tumor Cells

    doi: 10.3389/fonc.2014.00201

    Figure Lengend Snippet: Image-based capture of individual spiked cells . (A) Cell surface GD2 expression was measured by flow cytometry for the eight neuroblastoma cell lines used for isolation of single cells (and listed at right), as compared to negative control WBCs and isotype control. (B) WBCs and the GD2-dim neuroblastoma cell line SY5Y were mixed at a tumor:WBC ratio of 1:35, pre-labeled with GD2-PE, CD45-FITC, and Hoechst nuclear dye, then injected into the DEPArray. Shown are bright-field images of (i) a single tumor cell (bar depicts 10 μm) and (ii) a cluster of two cells. In (C) , a scatter plot of GD2-PE and CD45-FITC mean fluorescence intensity (MFI) is shown as well as cell images, including on the left-hand side of the dot-plot (i) an image of a single tumor cell and (ii) a single WBC, and on the right-hand side (iii) a heterogeneous cluster, (iv) a homogeneous cluster, and (v) a spurious event. (D) NB1643M cells and normal donor WBCs were mixed at a ratio of 1:1,000,000 and stained with GD2-PE and CD45-FITC, in this representative experiment. The sample was pre-enriched using FACSAria-based sorting, and the enriched fraction placed into the DEPArray cartridge. Shown are a scatter plot of GD2-PE and CD45-FITC MFI, as well as images of (i–iv) intact tumor cells, (v and vi) WBCs, and (vii–ix) debris and false-positive events.

    Article Snippet: Due to the 40,000 cell capacity of the DEPArray cartridge, pre-enrichment of the sample was first completed on the FACSAria II (Becton Dickinson Franklin Lakes, NJ, USA) prior to running on the DEPArray, if the tumor cell concentration was below 1 in 5,000 WBCs (0.02%).

    Techniques: Expressing, Flow Cytometry, Cytometry, Isolation, Negative Control, Labeling, Injection, Fluorescence, Staining

    Isolation and targeted sequencing of rare individual patient DTC . (A) The post-Ficoll fraction for a bone marrow sample from patient CHOP7 was fixed, stained for CD45, CD56, and GD2, and an aliquot was analyzed by flow cytometry. Given that only 0.02% of cells were determined to be GD2-positive, (B) pre-enrichment was conducted on the FACSAria using the wide P2 gate shown. (C) Representative images of staining for GD2 (first column; bar depicts 10 μm), CD56 (middle column), and CD45 (right column) are shown for single cells #15 (top row) and #7 (bottom row). (D) A gel for the quality control panel of four housekeeping genes is shown with a “+” below depicting detection of either the mutant allele (top row labeled “M” or the wild-type allele bottom row labeled “WT”). (E) The chromatogram of the wild-type (top) and mutant allele (bottom) for cell #8. (F) Diameter of patient cells, as measured for 56 tumor cells and 22 WBCs while in solution on the DEPArray chip. (G) Bone marrow samples of patients were processed as described above and direct fluorescent staining used to measure the DNA concentration of WGA product from single cells. Mean WGA yield for n = 64 patient single cells is shown in comparison to the yield for n = 31 single tumor cells from cell line spiking experiments. In this analysis, all patient and cell line cells were fixed.

    Journal: Frontiers in Oncology

    Article Title: Dielectrophoretic Capture and Genetic Analysis of Single Neuroblastoma Tumor Cells

    doi: 10.3389/fonc.2014.00201

    Figure Lengend Snippet: Isolation and targeted sequencing of rare individual patient DTC . (A) The post-Ficoll fraction for a bone marrow sample from patient CHOP7 was fixed, stained for CD45, CD56, and GD2, and an aliquot was analyzed by flow cytometry. Given that only 0.02% of cells were determined to be GD2-positive, (B) pre-enrichment was conducted on the FACSAria using the wide P2 gate shown. (C) Representative images of staining for GD2 (first column; bar depicts 10 μm), CD56 (middle column), and CD45 (right column) are shown for single cells #15 (top row) and #7 (bottom row). (D) A gel for the quality control panel of four housekeeping genes is shown with a “+” below depicting detection of either the mutant allele (top row labeled “M” or the wild-type allele bottom row labeled “WT”). (E) The chromatogram of the wild-type (top) and mutant allele (bottom) for cell #8. (F) Diameter of patient cells, as measured for 56 tumor cells and 22 WBCs while in solution on the DEPArray chip. (G) Bone marrow samples of patients were processed as described above and direct fluorescent staining used to measure the DNA concentration of WGA product from single cells. Mean WGA yield for n = 64 patient single cells is shown in comparison to the yield for n = 31 single tumor cells from cell line spiking experiments. In this analysis, all patient and cell line cells were fixed.

    Article Snippet: Due to the 40,000 cell capacity of the DEPArray cartridge, pre-enrichment of the sample was first completed on the FACSAria II (Becton Dickinson Franklin Lakes, NJ, USA) prior to running on the DEPArray, if the tumor cell concentration was below 1 in 5,000 WBCs (0.02%).

    Techniques: Isolation, Sequencing, Staining, Flow Cytometry, Cytometry, Mutagenesis, Labeling, Chromatin Immunoprecipitation, Concentration Assay, Whole Genome Amplification

    Workflow for rare single cell isolation by DEPArray . Shown are the steps for dielectrophoretic isolation and downstream genetic analysis of single cells. Because the DEPArray cartridge has a capacity of ~40,000 cells, if the tumor cell concentration is below 0.02%, pre-enrichment of the sample is first completed on the FACSAria.

    Journal: Frontiers in Oncology

    Article Title: Dielectrophoretic Capture and Genetic Analysis of Single Neuroblastoma Tumor Cells

    doi: 10.3389/fonc.2014.00201

    Figure Lengend Snippet: Workflow for rare single cell isolation by DEPArray . Shown are the steps for dielectrophoretic isolation and downstream genetic analysis of single cells. Because the DEPArray cartridge has a capacity of ~40,000 cells, if the tumor cell concentration is below 0.02%, pre-enrichment of the sample is first completed on the FACSAria.

    Article Snippet: Due to the 40,000 cell capacity of the DEPArray cartridge, pre-enrichment of the sample was first completed on the FACSAria II (Becton Dickinson Franklin Lakes, NJ, USA) prior to running on the DEPArray, if the tumor cell concentration was below 1 in 5,000 WBCs (0.02%).

    Techniques: Single-cell Isolation, Isolation, Concentration Assay

    Behaviour of an inverted ON switch generated via insertion of the module into an OFF switch. ( a ) Construction of the INPUT and OUTPUT plasmids transfected into HeLa cells in this study. The INPUT plasmids express either L7Ae or the MS2 coat protein (MS2) as a cognate or noncognate RNA-binding protein, respectively. DsRed-Express is also synthesized from the INPUT plasmid, driven by the IRES. An OUTPUT plasmid expresses ON-switch mRNAs containing K-turn (Kt) or defective K-turn (dKt) as either an active or defective sensory motif, respectively. EGFP driven by the IRES is synthesized from the OUTPUT plasmid. ( b – e ) Images of cells captured via fluorescence microscopy. DsRed-Express synthesized together with L7Ae ( c , e ) or MS2 ( b , d ) is shown in red. EGFP outputs from an ON switch with either an active ( b , c ; ON-Kt) or a defective ( d , e ; ON-dKt) sensor are shown in green. These two fluorescent signals are merged and shown in the right most pictures. Scale bars, 200 μm. ( f ) The mean intensity of EGFP fluorescence of transfected cells. HeLa cells shown in b – e were analysed using a flow cytometer, FACSAria. Cells expressing only DsRed-Express were used as the negative control (DsRed-Express). Bars and error bars represent the mean and s.d., respectively, of three triplicate experiments ( n =9). ( g ) The levels of ON-switch mRNAs, as determined by quantitative RT–PCR. To eliminate the noise from untransfected cells, the results are normalized by employing the mRNA of neomycin-resistant gene transcribed from the OUTPUT plasmid in cells. Bars and error bars represent the mean and s.d., respectively, of three triplicate experiments ( n =9). n.a., not analysed. All experiments in b – g were carried out 24 h after the transfection of 100 ng of the indicated OUTPUT plasmid and 20 ng of the INPUT plasmid together with 480 ng of the noncognate INPUT plasmid.

    Journal: Nature Communications

    Article Title: A versatile cis-acting inverter module for synthetic translational switches

    doi: 10.1038/ncomms3393

    Figure Lengend Snippet: Behaviour of an inverted ON switch generated via insertion of the module into an OFF switch. ( a ) Construction of the INPUT and OUTPUT plasmids transfected into HeLa cells in this study. The INPUT plasmids express either L7Ae or the MS2 coat protein (MS2) as a cognate or noncognate RNA-binding protein, respectively. DsRed-Express is also synthesized from the INPUT plasmid, driven by the IRES. An OUTPUT plasmid expresses ON-switch mRNAs containing K-turn (Kt) or defective K-turn (dKt) as either an active or defective sensory motif, respectively. EGFP driven by the IRES is synthesized from the OUTPUT plasmid. ( b – e ) Images of cells captured via fluorescence microscopy. DsRed-Express synthesized together with L7Ae ( c , e ) or MS2 ( b , d ) is shown in red. EGFP outputs from an ON switch with either an active ( b , c ; ON-Kt) or a defective ( d , e ; ON-dKt) sensor are shown in green. These two fluorescent signals are merged and shown in the right most pictures. Scale bars, 200 μm. ( f ) The mean intensity of EGFP fluorescence of transfected cells. HeLa cells shown in b – e were analysed using a flow cytometer, FACSAria. Cells expressing only DsRed-Express were used as the negative control (DsRed-Express). Bars and error bars represent the mean and s.d., respectively, of three triplicate experiments ( n =9). ( g ) The levels of ON-switch mRNAs, as determined by quantitative RT–PCR. To eliminate the noise from untransfected cells, the results are normalized by employing the mRNA of neomycin-resistant gene transcribed from the OUTPUT plasmid in cells. Bars and error bars represent the mean and s.d., respectively, of three triplicate experiments ( n =9). n.a., not analysed. All experiments in b – g were carried out 24 h after the transfection of 100 ng of the indicated OUTPUT plasmid and 20 ng of the INPUT plasmid together with 480 ng of the noncognate INPUT plasmid.

    Article Snippet: After addition of 100 μl of medium, the cells were passed through a 35 μm strainer (BD Biosciences, San Jose, CA) and then analysed with a FACSAria cell sorter (BD Biosciences) or BD Accuri (BD Biosciences).

    Techniques: Generated, Transfection, RNA Binding Assay, Synthesized, Plasmid Preparation, Fluorescence, Microscopy, Flow Cytometry, Cytometry, Expressing, Negative Control, Quantitative RT-PCR

    Induction of cell death using an inverted switch. ( a ) Schematic illustration of the experiment. Together with the INPUT plasmids (500 ng), we transfected two plasmids: one expressing anti-apoptotic gene, Bcl-xL (10 ng) and ON-Kt-B (or dKt, 100 ng), which outputs apoptotic gene, Bim-EL instead of EGFP. ( b ) Induction of annexin V-positive cells. For transfection, 20 ng of cognate and 480 ng of noncognate INPUT plasmids were mixed. The left panel shows histograms of Pacific Blue-labelled anti-annexin V using FACSAria. A black line indicates a threshold used in this analysis. The right panel shows a ratio of annexin V-positive cells to the transfected cells. Bars and error bars represent the mean and s.d., respectively, of two replicates.

    Journal: Nature Communications

    Article Title: A versatile cis-acting inverter module for synthetic translational switches

    doi: 10.1038/ncomms3393

    Figure Lengend Snippet: Induction of cell death using an inverted switch. ( a ) Schematic illustration of the experiment. Together with the INPUT plasmids (500 ng), we transfected two plasmids: one expressing anti-apoptotic gene, Bcl-xL (10 ng) and ON-Kt-B (or dKt, 100 ng), which outputs apoptotic gene, Bim-EL instead of EGFP. ( b ) Induction of annexin V-positive cells. For transfection, 20 ng of cognate and 480 ng of noncognate INPUT plasmids were mixed. The left panel shows histograms of Pacific Blue-labelled anti-annexin V using FACSAria. A black line indicates a threshold used in this analysis. The right panel shows a ratio of annexin V-positive cells to the transfected cells. Bars and error bars represent the mean and s.d., respectively, of two replicates.

    Article Snippet: After addition of 100 μl of medium, the cells were passed through a 35 μm strainer (BD Biosciences, San Jose, CA) and then analysed with a FACSAria cell sorter (BD Biosciences) or BD Accuri (BD Biosciences).

    Techniques: Transfection, Expressing

    Correlation of the sensitivity and reactivity between the inverted ON switches and parental OFF switches. ( a ) Behaviour of the switches as a function of the amount of input. The x a xis shows the ratio of the INPUT plasmids expressing cognate RNA-binding proteins to the OUTPUT plasmids for the indicated switches (100 ng). The total plasmid content was adjusted up to 600 ng using noncognate plasmids. Twenty-four hours after transfection, individual cells were analysed using FACSAria. Geometrical mean values of the ratio between EGFP and DsRed-Express signals in a cell were shown. ON-Fr15 and OFF-Fr15 are switches responsive to Bacillus ribosomal protein S15 instead of L7Ae. Data points and error bars represent the mean and s.d., respectively, of three replicates. See Supplementary Fig. S4 (ON-Kt and OFF-Kt) and Supplementary Fig. S5 (ON-Fr15 and OFF-Fr15) for plots produced from this analysis. ( b , c ) Western blotting analysis of input proteins is shown. Total protein from the transfected cells shown in a was extracted 24 h after transfection and subjected to western blotting analysis. Cognate (L7Ae ( b ) and S15 ( c )) and noncognate (MS2CP ( b ) and L7KK ( c )) input proteins were detected using anti-myc antibody. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was also analysed as an internal control of the lysates. The right three lanes were fivefold serial dilutions of the lysate prepared in the same condition as the ratio of INPUT plasmid/OUTPUT plasmid equal to 5. Representative result from the three independent experiments was shown. ( d ) Behaviour of the switches as a function of the affinity between the input protein and the corresponding sensory motif in the switch. See Supplementary Fig. S8 for plots generated from this analysis.

    Journal: Nature Communications

    Article Title: A versatile cis-acting inverter module for synthetic translational switches

    doi: 10.1038/ncomms3393

    Figure Lengend Snippet: Correlation of the sensitivity and reactivity between the inverted ON switches and parental OFF switches. ( a ) Behaviour of the switches as a function of the amount of input. The x a xis shows the ratio of the INPUT plasmids expressing cognate RNA-binding proteins to the OUTPUT plasmids for the indicated switches (100 ng). The total plasmid content was adjusted up to 600 ng using noncognate plasmids. Twenty-four hours after transfection, individual cells were analysed using FACSAria. Geometrical mean values of the ratio between EGFP and DsRed-Express signals in a cell were shown. ON-Fr15 and OFF-Fr15 are switches responsive to Bacillus ribosomal protein S15 instead of L7Ae. Data points and error bars represent the mean and s.d., respectively, of three replicates. See Supplementary Fig. S4 (ON-Kt and OFF-Kt) and Supplementary Fig. S5 (ON-Fr15 and OFF-Fr15) for plots produced from this analysis. ( b , c ) Western blotting analysis of input proteins is shown. Total protein from the transfected cells shown in a was extracted 24 h after transfection and subjected to western blotting analysis. Cognate (L7Ae ( b ) and S15 ( c )) and noncognate (MS2CP ( b ) and L7KK ( c )) input proteins were detected using anti-myc antibody. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was also analysed as an internal control of the lysates. The right three lanes were fivefold serial dilutions of the lysate prepared in the same condition as the ratio of INPUT plasmid/OUTPUT plasmid equal to 5. Representative result from the three independent experiments was shown. ( d ) Behaviour of the switches as a function of the affinity between the input protein and the corresponding sensory motif in the switch. See Supplementary Fig. S8 for plots generated from this analysis.

    Article Snippet: After addition of 100 μl of medium, the cells were passed through a 35 μm strainer (BD Biosciences, San Jose, CA) and then analysed with a FACSAria cell sorter (BD Biosciences) or BD Accuri (BD Biosciences).

    Techniques: Expressing, RNA Binding Assay, Plasmid Preparation, Transfection, Produced, Western Blot, Generated

    Simultaneous and symmetrical control of two outputs in a cell by using ON and OFF switches. ( a ) The behaviour of ON and OFF switches responsive to a single input (L7Ae). Two reporter proteins, EGFP and enhanced cyan fluorescent protein (ECFP), were produced from ON and OFF switches, respectively. Two OUTPUT plasmids expressing either an ON or an OFF switch (100 ng each) were transfected into HeLa cells together with 20 ng of cognate and 480 ng of noncognate INPUT plasmids. The fluorescent signals were normalized by those of the switches containing Kt in the absence of L7Ae. ( b ) Control of two switches under two input proteins. Kt or dKt in ON switches were replaced with Fr15 or dFr15 (defective Fr15). Two OUTPUT plasmids were transfected together with L7Ae- (20 ng) and/or S15- (480 ng) expressing plasmids. In this experiment, a viral nucleocapside protein was used as a noncognate RNA-binding protein. The fluorescent signals were normalized by those detected in the absence of both L7Ae and S15. Transfected cells were analysed using FACSAria. Bars and error bars represent the mean and s.d., respectively, of three replicates.

    Journal: Nature Communications

    Article Title: A versatile cis-acting inverter module for synthetic translational switches

    doi: 10.1038/ncomms3393

    Figure Lengend Snippet: Simultaneous and symmetrical control of two outputs in a cell by using ON and OFF switches. ( a ) The behaviour of ON and OFF switches responsive to a single input (L7Ae). Two reporter proteins, EGFP and enhanced cyan fluorescent protein (ECFP), were produced from ON and OFF switches, respectively. Two OUTPUT plasmids expressing either an ON or an OFF switch (100 ng each) were transfected into HeLa cells together with 20 ng of cognate and 480 ng of noncognate INPUT plasmids. The fluorescent signals were normalized by those of the switches containing Kt in the absence of L7Ae. ( b ) Control of two switches under two input proteins. Kt or dKt in ON switches were replaced with Fr15 or dFr15 (defective Fr15). Two OUTPUT plasmids were transfected together with L7Ae- (20 ng) and/or S15- (480 ng) expressing plasmids. In this experiment, a viral nucleocapside protein was used as a noncognate RNA-binding protein. The fluorescent signals were normalized by those detected in the absence of both L7Ae and S15. Transfected cells were analysed using FACSAria. Bars and error bars represent the mean and s.d., respectively, of three replicates.

    Article Snippet: After addition of 100 μl of medium, the cells were passed through a 35 μm strainer (BD Biosciences, San Jose, CA) and then analysed with a FACSAria cell sorter (BD Biosciences) or BD Accuri (BD Biosciences).

    Techniques: Produced, Expressing, Transfection, RNA Binding Assay