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  • 99
    Millipore flow cytometry flow cytometric analyses
    <t>Flow-cytometric</t> analysis of surface markers (CD11c, CD11b, and MHC class II) during generation of immature DCs from bone marrow cells in the presence of 10 ng/mL of granulocyte macrophage colony-stimulating factor on days 0, 3, and 7 of culture. Note: Numbers within dot plots represent percentages within the quadrant. Abbreviations: DCs, dendritic cells; MHC, major histocompatibility complex.
    Flow Cytometry Flow Cytometric Analyses, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 477 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher flow cytometry flow cytometric reference beads
    <t>Flow-cytometric</t> analysis of surface markers (CD11c, CD11b, and MHC class II) during generation of immature DCs from bone marrow cells in the presence of 10 ng/mL of granulocyte macrophage colony-stimulating factor on days 0, 3, and 7 of culture. Note: Numbers within dot plots represent percentages within the quadrant. Abbreviations: DCs, dendritic cells; MHC, major histocompatibility complex.
    Flow Cytometry Flow Cytometric Reference Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson flow cytometry a flow cytometric assay
    <t>Flow-cytometric</t> analysis of surface markers (CD11c, CD11b, and MHC class II) during generation of immature DCs from bone marrow cells in the presence of 10 ng/mL of granulocyte macrophage colony-stimulating factor on days 0, 3, and 7 of culture. Note: Numbers within dot plots represent percentages within the quadrant. Abbreviations: DCs, dendritic cells; MHC, major histocompatibility complex.
    Flow Cytometry A Flow Cytometric Assay, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore flow cytometry flow cytometry
    Generation of A549-derived cell-lines. A . Most stable mfold-predicted secondary structures of human pre-miR-146b and a variant pre-microRNA examined in this study. The 5′ and 3′ ends, nucleotide positions, and segments corresponding to mature miR-146b-5p and -3p sequences ( 5p and 3p ) are indicated. B . Histograms for red fluorescence quantified by flow <t>cytometry</t> of A549 and A549-derived cell-lines stably transduced with lentiviruses bearing constructs engineered for expression of human pre-miR-146b ( A549/146b ), or its variant shown in A ( A549/v146b ), or neither ( A549/vec ). C . Comparison of miR-146b-5p and -3p levels ( 5p and 3p ), normalized to that of the RNU6B small nucleolar RNA, in the A549-derived cell-lines as assessed using reverse transcription followed by PCR (RT-PCR). Means and their standard errors for measurements from three different experiments are shown. D . Standard curves showing the relationship between quantification cycle ( C q ) value and concentration of RNA in miR-146b-5p and -3p ( 5p and 3p ) RT-PCR assays of synthetic miR-146b-5p and -3p RNA, respectively. A log 2 scale is used for the X axis. E . Semi-quantification of the relative amounts of miR-146b-5p and -3p in RNA of 293T, BEAS-2B, MCF-7, and ML-2 (means, single experiments), and the three A549-derived cell-lines, (means and their standard errors for measurements from three different experiments) are shown.
    Flow Cytometry Flow Cytometry, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 581 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Miltenyi Biotec flow cytometry cytometric analysis
    Generation of A549-derived cell-lines. A . Most stable mfold-predicted secondary structures of human pre-miR-146b and a variant pre-microRNA examined in this study. The 5′ and 3′ ends, nucleotide positions, and segments corresponding to mature miR-146b-5p and -3p sequences ( 5p and 3p ) are indicated. B . Histograms for red fluorescence quantified by flow <t>cytometry</t> of A549 and A549-derived cell-lines stably transduced with lentiviruses bearing constructs engineered for expression of human pre-miR-146b ( A549/146b ), or its variant shown in A ( A549/v146b ), or neither ( A549/vec ). C . Comparison of miR-146b-5p and -3p levels ( 5p and 3p ), normalized to that of the RNU6B small nucleolar RNA, in the A549-derived cell-lines as assessed using reverse transcription followed by PCR (RT-PCR). Means and their standard errors for measurements from three different experiments are shown. D . Standard curves showing the relationship between quantification cycle ( C q ) value and concentration of RNA in miR-146b-5p and -3p ( 5p and 3p ) RT-PCR assays of synthetic miR-146b-5p and -3p RNA, respectively. A log 2 scale is used for the X axis. E . Semi-quantification of the relative amounts of miR-146b-5p and -3p in RNA of 293T, BEAS-2B, MCF-7, and ML-2 (means, single experiments), and the three A549-derived cell-lines, (means and their standard errors for measurements from three different experiments) are shown.
    Flow Cytometry Cytometric Analysis, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson flow cytometry flow cytometry
    Shorter shRNA length reduced, but did not eliminate, cytotoxicity. A represents flow <t>cytometry</t> analysis of IS-1 cells 4 and 10 days after transduction with U6PT, sh319, sh321, sh323 and sh325 vectors. GFP expression is detected, and the percentage of GFP expressing cells was determined using the M1 gating shown (percentage GFP positive cells is shown in each histogram). B shows a comparative analysis of PAI-2 and OAS1 mRNA, in the samples described in A, 4 days after transduction. Data were generated by QRT-PCR and error bars are as described in previous figures. In C, cell lysates from samples of transduced cells described in A and B were subjected to immunoblotting with anti-PAI-2 monoclonal antibodies. Ponceau S staining served as a gel loading control, as did comparison of a single non-specific band (NS) in the immunoblot. D shows QRT-PCR analysis, as in B, for IS-1 cell mRNAs after transduction with or without U6PT, sh319, sh319scr and sh321 vectors.
    Flow Cytometry Flow Cytometry, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 99/100, based on 1294 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson flow cytometric analysis flow cytometry abs
    Shorter shRNA length reduced, but did not eliminate, cytotoxicity. A represents flow <t>cytometry</t> analysis of IS-1 cells 4 and 10 days after transduction with U6PT, sh319, sh321, sh323 and sh325 vectors. GFP expression is detected, and the percentage of GFP expressing cells was determined using the M1 gating shown (percentage GFP positive cells is shown in each histogram). B shows a comparative analysis of PAI-2 and OAS1 mRNA, in the samples described in A, 4 days after transduction. Data were generated by QRT-PCR and error bars are as described in previous figures. In C, cell lysates from samples of transduced cells described in A and B were subjected to immunoblotting with anti-PAI-2 monoclonal antibodies. Ponceau S staining served as a gel loading control, as did comparison of a single non-specific band (NS) in the immunoblot. D shows QRT-PCR analysis, as in B, for IS-1 cell mRNAs after transduction with or without U6PT, sh319, sh319scr and sh321 vectors.
    Flow Cytometric Analysis Flow Cytometry Abs, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Flow-cytometric analysis of surface markers (CD11c, CD11b, and MHC class II) during generation of immature DCs from bone marrow cells in the presence of 10 ng/mL of granulocyte macrophage colony-stimulating factor on days 0, 3, and 7 of culture. Note: Numbers within dot plots represent percentages within the quadrant. Abbreviations: DCs, dendritic cells; MHC, major histocompatibility complex.

    Journal: Drug Design, Development and Therapy

    Article Title: Effects of histamine and its antagonists on murine T-cells and bone marrow-derived dendritic cells

    doi: 10.2147/DDDT.S89792

    Figure Lengend Snippet: Flow-cytometric analysis of surface markers (CD11c, CD11b, and MHC class II) during generation of immature DCs from bone marrow cells in the presence of 10 ng/mL of granulocyte macrophage colony-stimulating factor on days 0, 3, and 7 of culture. Note: Numbers within dot plots represent percentages within the quadrant. Abbreviations: DCs, dendritic cells; MHC, major histocompatibility complex.

    Article Snippet: Flow-cytometric analysis of murine spleen cells For in vitro stimulation, 2×106 spleen cells were mixed with 10 ng/mL of 4beta-phorbol 12-myristate 13-acetate (Sigma-Aldrich) and 1 µg/mL of ionomycin (Sigma-Aldrich) and incubated in a 24-well plate at 37°C and 5% CO2 .

    Techniques: Flow Cytometry

    Fraction of cells with decreased mitochondrial potential and increased [Ca 2+ ] i after Hemidesmus treatment. Alterations in mitochondrial membrane permeability in absence (A) and presence (B) of inhibitors following treatment with Hemidesmus 0.93 mg/mL. Fraction of cells with increased [Ca 2+ ] i following 3 or 24 h culture in the absence or presence of Hemidesmus (0.93 mg/mL) (C), flow cytometric analysis of [Ca 2+ ] i following 24 h culture in the absence or presence of Hemidesmus (0.93 mg/mL) (D). M1 and M2 indicate the two different populations characterized by a different mean fluorescence intensity values. Data are means ± SEM of four independent experiments.

    Journal: PLoS ONE

    Article Title: Mitochondrial Pathway Mediates the Antileukemic Effects of Hemidesmus Indicus, a Promising Botanical Drug

    doi: 10.1371/journal.pone.0021544

    Figure Lengend Snippet: Fraction of cells with decreased mitochondrial potential and increased [Ca 2+ ] i after Hemidesmus treatment. Alterations in mitochondrial membrane permeability in absence (A) and presence (B) of inhibitors following treatment with Hemidesmus 0.93 mg/mL. Fraction of cells with increased [Ca 2+ ] i following 3 or 24 h culture in the absence or presence of Hemidesmus (0.93 mg/mL) (C), flow cytometric analysis of [Ca 2+ ] i following 24 h culture in the absence or presence of Hemidesmus (0.93 mg/mL) (D). M1 and M2 indicate the two different populations characterized by a different mean fluorescence intensity values. Data are means ± SEM of four independent experiments.

    Article Snippet: Flow cytometry All flow cytometric procedures were performed with a Guava EasyCyte Mini flow cytometer (Guava Technologies-Millipore, Hayward, CA, USA).

    Techniques: Permeability, Flow Cytometry, Fluorescence

    ATM-depleted fibroblasts progressively accumulate ROS and oxidative protein damage. ( A ) Representative Western blot analysis on ATM-depleted fibroblasts showing upregulation of proteins involved in the antioxidant response. TIG1 cells were transfected with either a control siRNA (siCtrl) or an ATM-targeting siRNA (siATM#1, siATM #2). Actin was used as loading control. Densitometric quantification of the indicated proteins is reported at the bottom of the gel ( N = 2). ( B ) Western blot analysis on a representative time course depletion of ATM. TIG1 fibroblasts were treated as indicated and ATM expression was monitored in nuclear cell extracts. Lamin A/C was used as loading control. ( C ) Quantification of ROS content using flow-cytometry in siATM-treated fibroblasts. TIG1 fibroblasts were treated as indicated. H 2 O 2 (25 μM, 30 min) was used as a positive control for induction of ROS. ATM inhibitors (Ku-55933 – ATMi #1 and Ku-60019 – ATMi #2, both 10 μM) were provided fresh every 24 h for a total of 72 h ( N = 3). ( D ) Quantification of mitochondrial membrane potential (ΔΨm) in siATM-treated fibroblasts using flow-cytometry. Cells were treated as in panel C. Staurosporine (2 μM, 2 h) was used as a positive control for mitochondrial membrane depolarisation ( N = 3). ( E ) Detection of protein carbonylation in nuclear extracts obtained from fibroblasts depleted of ATM for the indicated time. Carbonylated proteins were detected by derivatisation with dinitrophenylhydrazone (DNP) followed by Western blot using an anti-DNP antibody. Equal amounts of cell extract were loaded in each lane. H 2 O 2 (1 mM, 30 minutes) was used as a positive control for induction of protein carbonylation. ( F ) Quantification of protein carbonylation in ATM-depleted fibroblasts; the histogram displays densitometric data from the analysis reported in panel E ( N = 6). Results are expressed as mean ± SD from the indicated number ( N ) of independent experiments: * P

    Journal: Nucleic Acids Research

    Article Title: Modulation of proteostasis counteracts oxidative stress and affects DNA base excision repair capacity in ATM-deficient cells

    doi: 10.1093/nar/gkx635

    Figure Lengend Snippet: ATM-depleted fibroblasts progressively accumulate ROS and oxidative protein damage. ( A ) Representative Western blot analysis on ATM-depleted fibroblasts showing upregulation of proteins involved in the antioxidant response. TIG1 cells were transfected with either a control siRNA (siCtrl) or an ATM-targeting siRNA (siATM#1, siATM #2). Actin was used as loading control. Densitometric quantification of the indicated proteins is reported at the bottom of the gel ( N = 2). ( B ) Western blot analysis on a representative time course depletion of ATM. TIG1 fibroblasts were treated as indicated and ATM expression was monitored in nuclear cell extracts. Lamin A/C was used as loading control. ( C ) Quantification of ROS content using flow-cytometry in siATM-treated fibroblasts. TIG1 fibroblasts were treated as indicated. H 2 O 2 (25 μM, 30 min) was used as a positive control for induction of ROS. ATM inhibitors (Ku-55933 – ATMi #1 and Ku-60019 – ATMi #2, both 10 μM) were provided fresh every 24 h for a total of 72 h ( N = 3). ( D ) Quantification of mitochondrial membrane potential (ΔΨm) in siATM-treated fibroblasts using flow-cytometry. Cells were treated as in panel C. Staurosporine (2 μM, 2 h) was used as a positive control for mitochondrial membrane depolarisation ( N = 3). ( E ) Detection of protein carbonylation in nuclear extracts obtained from fibroblasts depleted of ATM for the indicated time. Carbonylated proteins were detected by derivatisation with dinitrophenylhydrazone (DNP) followed by Western blot using an anti-DNP antibody. Equal amounts of cell extract were loaded in each lane. H 2 O 2 (1 mM, 30 minutes) was used as a positive control for induction of protein carbonylation. ( F ) Quantification of protein carbonylation in ATM-depleted fibroblasts; the histogram displays densitometric data from the analysis reported in panel E ( N = 6). Results are expressed as mean ± SD from the indicated number ( N ) of independent experiments: * P

    Article Snippet: Flow cytometry Determination of intracellular ROS content was carried out by staining with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (Sigma).

    Techniques: Western Blot, Transfection, Expressing, Flow Cytometry, Cytometry, Positive Control

    Anti-TNF treatment increases frequency of circulating T helper (Th)17 cells. Change in numbers of IL17-producing peripheral blood mononuclear cells during anti-TNF treatment determined by IL17 enzyme-linked immunospot (Elispot) assay ( a ) and representative IL17 Elispot experimental wells ( b ). PBMC (n = 200,000) were seeded in triplicate in each experiment and stimulated with 1 mg/ml anti-CD3 antibody for 20 hours and the numbers of cytokine-producing cells were enumerated. Change in the frequency of circulating CD4 + IL17+ cells in the peripheral blood of patients with rheumatoid arthritis (RA) determined by flow cytometry ( c ) and representative dot plots ( d ). Bars represent median and interquartile range; * p

    Journal: Arthritis Research & Therapy

    Article Title: Increase in circulating Th17 cells during anti-TNF therapy is associated with ultrasonographic improvement of synovitis in rheumatoid arthritis

    doi: 10.1186/s13075-016-1197-5

    Figure Lengend Snippet: Anti-TNF treatment increases frequency of circulating T helper (Th)17 cells. Change in numbers of IL17-producing peripheral blood mononuclear cells during anti-TNF treatment determined by IL17 enzyme-linked immunospot (Elispot) assay ( a ) and representative IL17 Elispot experimental wells ( b ). PBMC (n = 200,000) were seeded in triplicate in each experiment and stimulated with 1 mg/ml anti-CD3 antibody for 20 hours and the numbers of cytokine-producing cells were enumerated. Change in the frequency of circulating CD4 + IL17+ cells in the peripheral blood of patients with rheumatoid arthritis (RA) determined by flow cytometry ( c ) and representative dot plots ( d ). Bars represent median and interquartile range; * p

    Article Snippet: Flow cytometry quantitation of CD4 + IL17-producing cells Thawed PBMC at a concentration 15 × 106 /ml were placed in culture medium (RPMI 1640 supplemented with 10% FBS, 1% penicillin/streptomycin and 1% L-glutamine, all Sigma) and stimulated for 5 hours with phorbol myristate acetate (PMA, 50 ng/ml) and Ionomycin (500 ng/ml) (both Calbiochem, Nottingham, UK) in the presence of 10 μg/ml Brefeldin A (Sigma).

    Techniques: Enzyme-linked Immunospot, Flow Cytometry, Cytometry

    Generation of A549-derived cell-lines. A . Most stable mfold-predicted secondary structures of human pre-miR-146b and a variant pre-microRNA examined in this study. The 5′ and 3′ ends, nucleotide positions, and segments corresponding to mature miR-146b-5p and -3p sequences ( 5p and 3p ) are indicated. B . Histograms for red fluorescence quantified by flow cytometry of A549 and A549-derived cell-lines stably transduced with lentiviruses bearing constructs engineered for expression of human pre-miR-146b ( A549/146b ), or its variant shown in A ( A549/v146b ), or neither ( A549/vec ). C . Comparison of miR-146b-5p and -3p levels ( 5p and 3p ), normalized to that of the RNU6B small nucleolar RNA, in the A549-derived cell-lines as assessed using reverse transcription followed by PCR (RT-PCR). Means and their standard errors for measurements from three different experiments are shown. D . Standard curves showing the relationship between quantification cycle ( C q ) value and concentration of RNA in miR-146b-5p and -3p ( 5p and 3p ) RT-PCR assays of synthetic miR-146b-5p and -3p RNA, respectively. A log 2 scale is used for the X axis. E . Semi-quantification of the relative amounts of miR-146b-5p and -3p in RNA of 293T, BEAS-2B, MCF-7, and ML-2 (means, single experiments), and the three A549-derived cell-lines, (means and their standard errors for measurements from three different experiments) are shown.

    Journal: PLoS ONE

    Article Title: Overexpression of the Lung Cancer-Prognostic miR-146b MicroRNAs Has a Minimal and Negative Effect on the Malignant Phenotype of A549 Lung Cancer Cells

    doi: 10.1371/journal.pone.0022379

    Figure Lengend Snippet: Generation of A549-derived cell-lines. A . Most stable mfold-predicted secondary structures of human pre-miR-146b and a variant pre-microRNA examined in this study. The 5′ and 3′ ends, nucleotide positions, and segments corresponding to mature miR-146b-5p and -3p sequences ( 5p and 3p ) are indicated. B . Histograms for red fluorescence quantified by flow cytometry of A549 and A549-derived cell-lines stably transduced with lentiviruses bearing constructs engineered for expression of human pre-miR-146b ( A549/146b ), or its variant shown in A ( A549/v146b ), or neither ( A549/vec ). C . Comparison of miR-146b-5p and -3p levels ( 5p and 3p ), normalized to that of the RNU6B small nucleolar RNA, in the A549-derived cell-lines as assessed using reverse transcription followed by PCR (RT-PCR). Means and their standard errors for measurements from three different experiments are shown. D . Standard curves showing the relationship between quantification cycle ( C q ) value and concentration of RNA in miR-146b-5p and -3p ( 5p and 3p ) RT-PCR assays of synthetic miR-146b-5p and -3p RNA, respectively. A log 2 scale is used for the X axis. E . Semi-quantification of the relative amounts of miR-146b-5p and -3p in RNA of 293T, BEAS-2B, MCF-7, and ML-2 (means, single experiments), and the three A549-derived cell-lines, (means and their standard errors for measurements from three different experiments) are shown.

    Article Snippet: Flow cytometry Flow cytometry of freshly-harvested cells stained with 1 µg/ml of the 7 -amino-actinomycin D viability dye (Sigma®, St. Louis, MO) was performed on a FACSCalibur machine (BD Biosciences®, San Jose, CA).

    Techniques: Derivative Assay, Variant Assay, Fluorescence, Flow Cytometry, Cytometry, Stable Transfection, Transduction, Construct, Expressing, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Concentration Assay

    Existence of stem-like side population cells in the human retinoblastoma WERI-Rb1 cell line. The cells were stained with Hoechst 33,342 and sorted with flow cytometry.  A : A small population of cells (0.075±0.017%) was identified.  B : Addition of 50 μM verapamil markedly reduced the staining of the SP cells. (n=3; x-axis, Hoechst red fluorescence intensity; y-axis, Hoechst blue fluorescence intensity).

    Journal: Molecular Vision

    Article Title: Characterization and retinal neuron differentiation of WERI-Rb1 cancer stem cells

    doi:

    Figure Lengend Snippet: Existence of stem-like side population cells in the human retinoblastoma WERI-Rb1 cell line. The cells were stained with Hoechst 33,342 and sorted with flow cytometry. A : A small population of cells (0.075±0.017%) was identified. B : Addition of 50 μM verapamil markedly reduced the staining of the SP cells. (n=3; x-axis, Hoechst red fluorescence intensity; y-axis, Hoechst blue fluorescence intensity).

    Article Snippet: CD133 staining for flow cytometry A sample of 1×107 cells in suspension was collected, and the cells were resuspended in PBS with 0.5% BSA (BSA; Sigma-Aldrich) and 2 mM EDTA (Sigma-Aldrich).

    Techniques: Staining, Flow Cytometry, Cytometry, Fluorescence

    Cytometry analysis of htMSCs . Panel A) Analyzed markers, its commitment, its expression (positive or negative) in fresh digested hFTs and in htMSCs, and the mean percentage of positive labeled cells and analyzed by flow cytometry (GuavaTechnologies, Hayward, CA, ). NP means

    Journal: Journal of Translational Medicine

    Article Title: Human fallopian tube: a new source of multipotent adult mesenchymal stem cells discarded in surgical procedures

    doi: 10.1186/1479-5876-7-46

    Figure Lengend Snippet: Cytometry analysis of htMSCs . Panel A) Analyzed markers, its commitment, its expression (positive or negative) in fresh digested hFTs and in htMSCs, and the mean percentage of positive labeled cells and analyzed by flow cytometry (GuavaTechnologies, Hayward, CA, ). NP means "not performed". Panel B) Related graphs, where it is possible to compare, for each of the 19 analyzed markers, the control sample (not labeled htMSCs) in gray and the experimental population of htMSCs (labeled with specific antibodies) in black.

    Article Snippet: Flow Cytometry Analysis Flow cytometry analysis was performed using a Guava EasyCyte microcapillary flow cytometer (Guava Technologies, Hayward, CA) utilizing laser excitation and emission wavelengths of 488 and 532 nm, respectively.

    Techniques: Cytometry, Expressing, Labeling, Flow Cytometry

    (a) Confocal fluorescence microscopy (CFM) images of KB and A549 cells after treated with different GNPs. All GNPs were labeled with TA-FITC which had green fluorescence. The scale bar is 50 μm. Flow cytometry assay to monitor the binding of GNPs

    Journal: Biomaterials

    Article Title: Enhancement of cell recognition in vitro by dual-ligand cancer targeting gold naoparticles

    doi: 10.1016/j.biomaterials.2010.12.031

    Figure Lengend Snippet: (a) Confocal fluorescence microscopy (CFM) images of KB and A549 cells after treated with different GNPs. All GNPs were labeled with TA-FITC which had green fluorescence. The scale bar is 50 μm. Flow cytometry assay to monitor the binding of GNPs

    Article Snippet: Flow cytometry analyses were performed on a Guava EasyCyte Mini flow cytometry system (Millipore, Billerica, MA).

    Techniques: Fluorescence, Microscopy, Labeling, Flow Cytometry, Cytometry, Binding Assay

    SOX2 and NeuN are co-expressed in the human dentate gyrus. ( a ) Representative images of human hippocampus sections showing immunofluorescence staining for SOX2 (red) and NeuN (green) in the dentate gyrus. Nuclei are counterstained blue with DAPI. Arrowheads indicate cells that co-express SOX2 and NeuN. ( b ) Immunofluorescent staining of SOX2 (red) and NeuN (green) in wild type mouse hippocampus dentate gyrus. Nuclei are stained blue with DAPI. ( c ) Representative Western blot showing human hippocampus lysate probed with the same anti-SOX2 antibody used in A and B. A band corresponding to the predicted molecular weight for SOX2 (34 kDa) is detected. ( d ) Flow cytometry analysis of NeuN and SOX2 expression in nuclei isolated from mouse and human hippocampus. Data is expressed as percentage of nuclei co-expressing SOX2 and NeuN versus total neuronal nuclei (expressing NeuN).

    Journal: Scientific Reports

    Article Title: Preserved neurogenesis in non-demented individuals with AD neuropathology

    doi: 10.1038/srep27812

    Figure Lengend Snippet: SOX2 and NeuN are co-expressed in the human dentate gyrus. ( a ) Representative images of human hippocampus sections showing immunofluorescence staining for SOX2 (red) and NeuN (green) in the dentate gyrus. Nuclei are counterstained blue with DAPI. Arrowheads indicate cells that co-express SOX2 and NeuN. ( b ) Immunofluorescent staining of SOX2 (red) and NeuN (green) in wild type mouse hippocampus dentate gyrus. Nuclei are stained blue with DAPI. ( c ) Representative Western blot showing human hippocampus lysate probed with the same anti-SOX2 antibody used in A and B. A band corresponding to the predicted molecular weight for SOX2 (34 kDa) is detected. ( d ) Flow cytometry analysis of NeuN and SOX2 expression in nuclei isolated from mouse and human hippocampus. Data is expressed as percentage of nuclei co-expressing SOX2 and NeuN versus total neuronal nuclei (expressing NeuN).

    Article Snippet: Immunolabeling and flow cytometry analysis of NeuN and SOX2 For flow cytometry analysis of NeuN and SOX2, the nuclei were spun down at 10,000 g at 4 °C for 5 min and resuspended in sodium citrate buffer (1 mg/ml sodium citrate, 1% Triton-X100 in Ca2+ and Mg2+ free PBS) containing RNase A (Sigma-Aldrich, MO) to prevent clumping of the nuclei.

    Techniques: Immunofluorescence, Staining, Western Blot, Molecular Weight, Flow Cytometry, Cytometry, Expressing, Isolation

    Purity verification of separated CD4 + T cells by flow cytometry. (a) showed the isotype control performed by Simultest IgG2a/IgG1. (b) represents double staining by anti-human CD3 and CD4 antibody. (c) represents anti-human antibody CD4 staining. These results showed that the purity of CD4 + from PBMC can reach a percent of more than 95%.

    Journal: Mediators of Inflammation

    Article Title: Clinical Significance of IL-23 Regulating IL-17A and/or IL-17F Positive Th17 Cells in Chronic Periodontitis

    doi: 10.1155/2014/627959

    Figure Lengend Snippet: Purity verification of separated CD4 + T cells by flow cytometry. (a) showed the isotype control performed by Simultest IgG2a/IgG1. (b) represents double staining by anti-human CD3 and CD4 antibody. (c) represents anti-human antibody CD4 staining. These results showed that the purity of CD4 + from PBMC can reach a percent of more than 95%.

    Article Snippet: Flow Cytometry Detection of IL-17A+ and/or IL-17F+ Th17 Cells After stimulation with rhIL-23 for 24 hours, cells were cultured in the presence of 50 ng/mL phorbol 12-myristate 13-acetate (P1585, Sigma St. Louis, MO, USA) and 500 ng/mL ionomycin (I0634, Sigma, USA) and with the protein transport inhibitor BD Golgistop 0.7 μ L/mL (Cat. number 554714, BD Bioscience) to prevent the cytokine secretion and allow cytokine accumulation inside the cell.

    Techniques: Flow Cytometry, Cytometry, Double Staining, Staining

    Evaluation of AP contribution to adipogenesis in mgWAT. a , b Representative flow cytometry plots of CD31−/CD45− (Lin−), CD34+, CD29+, Sca-1+, CD24± populations from the stromal vascular fraction (SVF) of fixed murine inguinal/mgWAT at involution day 1 ( a , left panel) or CD45−, CD34+, CD90+ cells within the SVF of human breast tissue ( b , left panel). Representative images of unpurified cells in total SVF or FACS purified adipocyte precursors (APs) from mice ( a , right panel) or humans ( b , right panel) after culturing in adipogenic media for 8 days ( a ) or 13 days ( b ) and staining with Oil Red O. n = 3 biological repeats. c Schematic summarizing stages of adipogenesis. d Experimental strategy to measure EdU incorporation in adipocyte precursors by FACS (as in e and f ), and in mature adipocytes (as in g , h ). e Representative flow cytometry plots of EdU signal in APs (as defined in a ) in virgin mice and at involution days 1 and 3. f The average percentage of EdU+ APs identified with flow cytometry as in e . n = 4 mice per time point, P = 0.51 for virgin vs. involution day 1 data. g, h Representative image ( g ) and quantification ( h ) of EdU immunofluorescence in Adi p oq-Cre; mT/mG mouse injected with EdU according to the timeline in d . Note EdU+ (arrow) and EdU− nuclei (arrowhead) in mGFP+ adipocytes. White box indicates insets. n = 200–800 nuclei in each of four mice per group, P = 0.84. i Experimental strategy to inhibit adipogenesis during involution using GW9662, a pharmacological antagonist of PPARγ. j , k Representative image ( j ) and quantification ( k ) of MGs from GW9662- and vehicle-treated mice, stained with a perilipin antibody and DAPI. N = 8–17 fields in each of five mice per treatment group, P = 0.09. Scale bar is 50 μm in g and j . Data are mean ± SEM. Statistics were performed using a one-way ANOVA with Tukey’s multiple comparisons test ( f ) and a two-tailed unpaired t -test ( h , k ). **** P

    Journal: Nature Communications

    Article Title: Adipocyte hypertrophy and lipid dynamics underlie mammary gland remodeling after lactation

    doi: 10.1038/s41467-018-05911-0

    Figure Lengend Snippet: Evaluation of AP contribution to adipogenesis in mgWAT. a , b Representative flow cytometry plots of CD31−/CD45− (Lin−), CD34+, CD29+, Sca-1+, CD24± populations from the stromal vascular fraction (SVF) of fixed murine inguinal/mgWAT at involution day 1 ( a , left panel) or CD45−, CD34+, CD90+ cells within the SVF of human breast tissue ( b , left panel). Representative images of unpurified cells in total SVF or FACS purified adipocyte precursors (APs) from mice ( a , right panel) or humans ( b , right panel) after culturing in adipogenic media for 8 days ( a ) or 13 days ( b ) and staining with Oil Red O. n = 3 biological repeats. c Schematic summarizing stages of adipogenesis. d Experimental strategy to measure EdU incorporation in adipocyte precursors by FACS (as in e and f ), and in mature adipocytes (as in g , h ). e Representative flow cytometry plots of EdU signal in APs (as defined in a ) in virgin mice and at involution days 1 and 3. f The average percentage of EdU+ APs identified with flow cytometry as in e . n = 4 mice per time point, P = 0.51 for virgin vs. involution day 1 data. g, h Representative image ( g ) and quantification ( h ) of EdU immunofluorescence in Adi p oq-Cre; mT/mG mouse injected with EdU according to the timeline in d . Note EdU+ (arrow) and EdU− nuclei (arrowhead) in mGFP+ adipocytes. White box indicates insets. n = 200–800 nuclei in each of four mice per group, P = 0.84. i Experimental strategy to inhibit adipogenesis during involution using GW9662, a pharmacological antagonist of PPARγ. j , k Representative image ( j ) and quantification ( k ) of MGs from GW9662- and vehicle-treated mice, stained with a perilipin antibody and DAPI. N = 8–17 fields in each of five mice per treatment group, P = 0.09. Scale bar is 50 μm in g and j . Data are mean ± SEM. Statistics were performed using a one-way ANOVA with Tukey’s multiple comparisons test ( f ) and a two-tailed unpaired t -test ( h , k ). **** P

    Article Snippet: Flow cytometry sorting and analysis For flow cytometry of primary murine cells, MG or skin tissue was minced and digested in Hanks Balanced Salt Solution (HBSS) (Sigma, H8264) supplemented with 3% BSA, 0.8 mg/ml collagenase type 2 (Worthington Biochemical, LS004174), 0.8 mM ZnCl2 , 1.0 mM MgCl2 and 1.2 mM CaCl2 for 75 min at 37 °C in a shaking water bath.

    Techniques: Flow Cytometry, Cytometry, FACS, Purification, Mouse Assay, Staining, Immunofluorescence, Injection, Two Tailed Test

    Orthotopic in vivo implantation of CXCR4 overexpressing cells reveals their tumorigenic potential and their intrinsic aggressiveness. A) Schematic representation of lentivirus coding for CXCR4 or a control gene. B) Cytometry analysis performed on U87MG transfected cells. The 12G5 antibody efficiently labeled U87MG CXCR4+ cells but not U87MG negative cells, transfected with the control lentivirus. C) Representative MRI performed at day 9 and day 17 after implantation of U87 CXCR4- cells and U87 CXCR4+ cells (arrows indicate tumor tissue) with resulting apparent tumor volumes and corresponding Kaplan-Meier curves. D) Immunohistochemistry analysis of U87 CXCR4+ tumors 12 days after implantation. CXCR4, CD31, CD11b and corresponding isotype control labeling appears in green (arrows) with RFP in red.

    Journal: Theranostics

    Article Title: Locoregional Confinement and Major Clinical Benefit of 188Re-Loaded CXCR4-Targeted Nanocarriers in an Orthotopic Human to Mouse Model of Glioblastoma

    doi: 10.7150/thno.19403

    Figure Lengend Snippet: Orthotopic in vivo implantation of CXCR4 overexpressing cells reveals their tumorigenic potential and their intrinsic aggressiveness. A) Schematic representation of lentivirus coding for CXCR4 or a control gene. B) Cytometry analysis performed on U87MG transfected cells. The 12G5 antibody efficiently labeled U87MG CXCR4+ cells but not U87MG negative cells, transfected with the control lentivirus. C) Representative MRI performed at day 9 and day 17 after implantation of U87 CXCR4- cells and U87 CXCR4+ cells (arrows indicate tumor tissue) with resulting apparent tumor volumes and corresponding Kaplan-Meier curves. D) Immunohistochemistry analysis of U87 CXCR4+ tumors 12 days after implantation. CXCR4, CD31, CD11b and corresponding isotype control labeling appears in green (arrows) with RFP in red.

    Article Snippet: Flow cytometry U87MG were collected and dissociated using trypsin (Sigma-Aldrich).

    Techniques: In Vivo, Cytometry, Transfection, Labeling, Magnetic Resonance Imaging, Immunohistochemistry

    Very delayed rPostC reverses post-stroke peripheral immunosuppression. Flow cytometry analysis from blood samples was performed 7 days after stroke induction in mice that had received rPostC 120 h after stroke or in control mice that had not received rPostC (non-rPostC). Sham animals underwent surgical procedure for transient focal cerebral ischemia without inserting of the filament into the left middle cerebral artery. A quantitative analysis of leukocytes (A) , neutrophils (B) , B-lymphocytes (C) , and T-lymphocytes (D) was performed as described in the materials and methods section. ∗ Significantly different from non-rPostC, ( p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Very Delayed Remote Ischemic Post-conditioning Induces Sustained Neurological Recovery by Mechanisms Involving Enhanced Angioneurogenesis and Peripheral Immunosuppression Reversal

    doi: 10.3389/fncel.2018.00383

    Figure Lengend Snippet: Very delayed rPostC reverses post-stroke peripheral immunosuppression. Flow cytometry analysis from blood samples was performed 7 days after stroke induction in mice that had received rPostC 120 h after stroke or in control mice that had not received rPostC (non-rPostC). Sham animals underwent surgical procedure for transient focal cerebral ischemia without inserting of the filament into the left middle cerebral artery. A quantitative analysis of leukocytes (A) , neutrophils (B) , B-lymphocytes (C) , and T-lymphocytes (D) was performed as described in the materials and methods section. ∗ Significantly different from non-rPostC, ( p

    Article Snippet: For flow cytometry measurements of brain tissue samples, ischemic left hemispheres were mechanically homogenized in a buffer of collagenase type XI (125 U/ml), hyaluronidase (60 U/ml) and collagenase (450 U/ml) in Ca2+ /Mg2+ supplemented PBS (Sigma-Aldrich, Germany).

    Techniques: Flow Cytometry, Cytometry, Mouse Assay

    Internalization and binding analysis of the H103 scFv Ab ( A ) Normoxic or hypoxicc treated HCCLM3 cells were incubated with H103 scFv Ab either in phage-display form or soluble form. After washing with PBST, the binding and uptake of the H103 scFv Ab was detected with AF488 conjugated anti-M13 (PVIII) or anti-His Abs under a confocal microscope. E4B7S scFv was used as the control. ( B ) The uptakes of the H103 scFv Ab at different time points in hypoxic HCCLM3 cells were measured by flow cytometric analysis. ( C ) After detachment with PBS/EDTA or T/E, the binding of the H103 scFv Ab on HCCLM3 cells was analyzed by flow cytometry.

    Journal: Oncotarget

    Article Title: Phage display library selection of a hypoxia-binding scFv antibody for liver cancer metabolic marker discovery

    doi: 10.18632/oncotarget.9460

    Figure Lengend Snippet: Internalization and binding analysis of the H103 scFv Ab ( A ) Normoxic or hypoxicc treated HCCLM3 cells were incubated with H103 scFv Ab either in phage-display form or soluble form. After washing with PBST, the binding and uptake of the H103 scFv Ab was detected with AF488 conjugated anti-M13 (PVIII) or anti-His Abs under a confocal microscope. E4B7S scFv was used as the control. ( B ) The uptakes of the H103 scFv Ab at different time points in hypoxic HCCLM3 cells were measured by flow cytometric analysis. ( C ) After detachment with PBS/EDTA or T/E, the binding of the H103 scFv Ab on HCCLM3 cells was analyzed by flow cytometry.

    Article Snippet: The final sub-library in each round was rescued again and subjected to the next round of panning. (2) Flow cytometry-based clone screening: phage-scFv and the soluble scFv Ab (periplasmic extract of scFv-phage-infected HB2151 E. coli cells) from individual clones were rocking-incubated with hypoxic cells (PBS/EDTA detached) at 4°C for 1 h. Bound phage scFv Abs were detected by adding a mixture of biotinylated anti-M13 McAb (1:600, Sigma) and streptavidin-AF488 (1:1000, Molecular Probes).

    Techniques: Binding Assay, Incubation, Microscopy, Flow Cytometry, Cytometry

    Inhibition of Axl restores ponatinib sensitivity in the K562-R and K562 DOX-R cell lines while AXL knockdown increases ponatinib sensitivity (A, B) Viability as assessed by Flow cytometry (AnnexinV-PE/7AAD double negative). The presence of the Axl inhibitor R428 (1 μM) or BMS777607 (12.5 μM) induced cell death significantly in (A) K562-R and (B) K562 DOX-R when co-treated with 200 nM ponatinib compared to their corresponding control lines. (C, D) Cell surface Axl expression was measured by flow cytometry in the AXL or scramble control shRNA transduced resistant cell lines. Compared to scramble control, AXL shRNA transduced cells demonstrated a reduction in Axl expression on the cell surface. (E, F) AXL knockdown in the K562 and K562 DOX ponatinib resistant cell lines demonstrated re-sensitisation to 10 nM ponatinib. Error bars represent SD, n≥3. * p

    Journal: Oncotarget

    Article Title: Modelling ponatinib resistance in tyrosine kinase inhibitor-naïve and dasatinib resistant BCR-ABL1+ cell lines

    doi: 10.18632/oncotarget.26187

    Figure Lengend Snippet: Inhibition of Axl restores ponatinib sensitivity in the K562-R and K562 DOX-R cell lines while AXL knockdown increases ponatinib sensitivity (A, B) Viability as assessed by Flow cytometry (AnnexinV-PE/7AAD double negative). The presence of the Axl inhibitor R428 (1 μM) or BMS777607 (12.5 μM) induced cell death significantly in (A) K562-R and (B) K562 DOX-R when co-treated with 200 nM ponatinib compared to their corresponding control lines. (C, D) Cell surface Axl expression was measured by flow cytometry in the AXL or scramble control shRNA transduced resistant cell lines. Compared to scramble control, AXL shRNA transduced cells demonstrated a reduction in Axl expression on the cell surface. (E, F) AXL knockdown in the K562 and K562 DOX ponatinib resistant cell lines demonstrated re-sensitisation to 10 nM ponatinib. Error bars represent SD, n≥3. * p

    Article Snippet: Immunophenotyping by Flow Cytometry: measurement of protein cell surface expression 5 × 105 cells were incubated with Hanks Balanced Salt Solution (HBSS) (JRH Biosciences, Australia) supplemented with 10mM Hepes (Sigma-Aldrich) and with corresponding isotype controls: IgG2aPE/IgG2bPE (Dako, Australia), IgG1PE (BD Biosciences, Australia); or PE-conjugated anti-hBCRP1 (ABCG2-PE) (R & D Systems, Australia), PE-conjugated anti-CD243 (ABCB1-PE) (Beckman Coulter, Australia), PE-conjugated anti-hAxl (R & D Systems, Australia) or PE-conjugated anti-hCD44 (BD Biosciences, Australia) antibodies for 40 minutes on ice.

    Techniques: Inhibition, Flow Cytometry, Cytometry, Expressing, shRNA

    Resistant cell lines K562-R and K562 DOX-R demonstrate increased adhesion compared to their corresponding control lines (A) Adherent cells were observed in both resistant cell lines, but not in their respective control lines, by light microscopy with 20X magnification. (B) Cells from resistant and control cultures were analysed by flow cytometry for expression of the adhesion marker CD44 (CD44-PE green; unstained grey; IgG2b PE isotype control red).

    Journal: Oncotarget

    Article Title: Modelling ponatinib resistance in tyrosine kinase inhibitor-naïve and dasatinib resistant BCR-ABL1+ cell lines

    doi: 10.18632/oncotarget.26187

    Figure Lengend Snippet: Resistant cell lines K562-R and K562 DOX-R demonstrate increased adhesion compared to their corresponding control lines (A) Adherent cells were observed in both resistant cell lines, but not in their respective control lines, by light microscopy with 20X magnification. (B) Cells from resistant and control cultures were analysed by flow cytometry for expression of the adhesion marker CD44 (CD44-PE green; unstained grey; IgG2b PE isotype control red).

    Article Snippet: Immunophenotyping by Flow Cytometry: measurement of protein cell surface expression 5 × 105 cells were incubated with Hanks Balanced Salt Solution (HBSS) (JRH Biosciences, Australia) supplemented with 10mM Hepes (Sigma-Aldrich) and with corresponding isotype controls: IgG2aPE/IgG2bPE (Dako, Australia), IgG1PE (BD Biosciences, Australia); or PE-conjugated anti-hBCRP1 (ABCG2-PE) (R & D Systems, Australia), PE-conjugated anti-CD243 (ABCB1-PE) (Beckman Coulter, Australia), PE-conjugated anti-hAxl (R & D Systems, Australia) or PE-conjugated anti-hCD44 (BD Biosciences, Australia) antibodies for 40 minutes on ice.

    Techniques: Light Microscopy, Flow Cytometry, Cytometry, Expressing, Marker

    SLAMF5-mediated enhancement of dendritic cell autophagy under steady-state conditions and in response to LPS/IFNγ activation. Control and SLAMF5 -silenced moDCs were stimulated or not with LPS/IFNγ for the indicated time periods in the absence [ (A) cropped] or in the presence of 20 nM bafilomycin A1 (BafA1) applied for the last 2 h (B) . Conversion of LC3 was determined by western blotting. A representative immunoblot of four independent experiments is shown on the left; ratios of LC3-II and β-actin determined by densitometry are shown in the right panels. (C) Control and SLAMF5-depleted moDCs were stained with CYTO-ID. Fluorescence intensity was determined by flow cytometry. Graph depicts the relative fluorescence intensity of CYTO-ID obtained in four independent experiments (D,E) . moDCs were conditioned with 10 μg ml −1 SLAMF5-specific or control antibodies followed by cross-linking with 10 μg ml −1 F(ab′) 2 fragment of goat anti-mouse IgG, then stimulated with LPS/IFNγ for 8 h in the absence (D) or in the presence (E) of 20 nM BafA1 applied for the last 2 h of the experiment. LC3 and β-actin levels were analyzed by western blotting; a representative blot and the mean ratios of LC3-II to β-actin from three independent experiments are shown. Data are expressed as the mean ± SD (* p

    Journal: Frontiers in Immunology

    Article Title: Signaling Lymphocyte Activation Molecule Family 5 Enhances Autophagy and Fine-Tunes Cytokine Response in Monocyte-Derived Dendritic Cells via Stabilization of Interferon Regulatory Factor 8

    doi: 10.3389/fimmu.2018.00062

    Figure Lengend Snippet: SLAMF5-mediated enhancement of dendritic cell autophagy under steady-state conditions and in response to LPS/IFNγ activation. Control and SLAMF5 -silenced moDCs were stimulated or not with LPS/IFNγ for the indicated time periods in the absence [ (A) cropped] or in the presence of 20 nM bafilomycin A1 (BafA1) applied for the last 2 h (B) . Conversion of LC3 was determined by western blotting. A representative immunoblot of four independent experiments is shown on the left; ratios of LC3-II and β-actin determined by densitometry are shown in the right panels. (C) Control and SLAMF5-depleted moDCs were stained with CYTO-ID. Fluorescence intensity was determined by flow cytometry. Graph depicts the relative fluorescence intensity of CYTO-ID obtained in four independent experiments (D,E) . moDCs were conditioned with 10 μg ml −1 SLAMF5-specific or control antibodies followed by cross-linking with 10 μg ml −1 F(ab′) 2 fragment of goat anti-mouse IgG, then stimulated with LPS/IFNγ for 8 h in the absence (D) or in the presence (E) of 20 nM BafA1 applied for the last 2 h of the experiment. LC3 and β-actin levels were analyzed by western blotting; a representative blot and the mean ratios of LC3-II to β-actin from three independent experiments are shown. Data are expressed as the mean ± SD (* p

    Article Snippet: Flow Cytometry Viability of the cells was determined on day 5 by 7-aminoactinomycin-D (7-AAD 10 µg ml−1 ; Sigma-Aldrich) staining for 15 min immediately before flow cytometry.

    Techniques: Activation Assay, Western Blot, Staining, Fluorescence, Flow Cytometry, Cytometry

    SLAMF5 controls the level of IRF8 protein, a positive regulator of autophagy in moDCs. (A) moDCs transfected with either control or an SLAMF5 -specific siRNA were treated with LPS (100 ng ml −1 ) and IFNγ (10 ng ml −1 ) for the indicated time periods. IRF8 protein level was measured by immunoblotting, and the ratio of IRF8/β-actin was quantified from five independent experiments. (B) Efficiency of IRF8 knockdown was tested on day 5 by western blot analysis; a representative blot is shown with β-actin as loading control. (C) Expression levels of CD14 and DC-SIGN as well as CD1a (D) were measured in control and IRF8 knockdown moDCs by flow cytometry. Bars show the relative fluorescence intensity values or the percentage of positive cells. The results shown are taken from three independent experiments. LC3-II expression was measured in control or IRF8 -silenced moDCs by immunoblotting in the absence (E) or in the presence of bafilomycin A1 (BafA1) (F) . One representative of three experiments is shown. Bar charts display the ratio of LC3-II/β-actin. (G) CYTO-ID staining of control and IRF8 -silenced moDCs from three donors was analyzed by flow cytometry. (H) CYTO-ID staining of control and IRF8 -silenced moDCs in which SLAMF5 was cross-linked with the SLAMF5-specific agonistic antibody 152.1D5 as described in Section “ Materials and Methods .” Data are expressed as the mean ± SD (* p

    Journal: Frontiers in Immunology

    Article Title: Signaling Lymphocyte Activation Molecule Family 5 Enhances Autophagy and Fine-Tunes Cytokine Response in Monocyte-Derived Dendritic Cells via Stabilization of Interferon Regulatory Factor 8

    doi: 10.3389/fimmu.2018.00062

    Figure Lengend Snippet: SLAMF5 controls the level of IRF8 protein, a positive regulator of autophagy in moDCs. (A) moDCs transfected with either control or an SLAMF5 -specific siRNA were treated with LPS (100 ng ml −1 ) and IFNγ (10 ng ml −1 ) for the indicated time periods. IRF8 protein level was measured by immunoblotting, and the ratio of IRF8/β-actin was quantified from five independent experiments. (B) Efficiency of IRF8 knockdown was tested on day 5 by western blot analysis; a representative blot is shown with β-actin as loading control. (C) Expression levels of CD14 and DC-SIGN as well as CD1a (D) were measured in control and IRF8 knockdown moDCs by flow cytometry. Bars show the relative fluorescence intensity values or the percentage of positive cells. The results shown are taken from three independent experiments. LC3-II expression was measured in control or IRF8 -silenced moDCs by immunoblotting in the absence (E) or in the presence of bafilomycin A1 (BafA1) (F) . One representative of three experiments is shown. Bar charts display the ratio of LC3-II/β-actin. (G) CYTO-ID staining of control and IRF8 -silenced moDCs from three donors was analyzed by flow cytometry. (H) CYTO-ID staining of control and IRF8 -silenced moDCs in which SLAMF5 was cross-linked with the SLAMF5-specific agonistic antibody 152.1D5 as described in Section “ Materials and Methods .” Data are expressed as the mean ± SD (* p

    Article Snippet: Flow Cytometry Viability of the cells was determined on day 5 by 7-aminoactinomycin-D (7-AAD 10 µg ml−1 ; Sigma-Aldrich) staining for 15 min immediately before flow cytometry.

    Techniques: Transfection, Western Blot, Expressing, Flow Cytometry, Cytometry, Fluorescence, Staining

    The effect of SLAMF5 silencing on the phenotype of moDCs. (A) Cell surface expression of SLAMF5 on monocytes and on GM-CSF/IL-4-differentiated moDCs treated or not with LPS/IFNγ was assessed by flow cytometry. (B) Monocytes were transfected with the indicated siRNAs and differentiated into moDCs. On day 5, SLAMF5 expression was measured by flow cytometry and western blot analysis. β-actin was used as protein loading control. (C) Viability of control and SLAMF5 knockdown moDCs was determined by 7-aminoactinomycin-D (7-AAD) dye exclusion. Bar graphs indicate percentage of cells negative for 7-AAD (left panel) and the total number of moDCs differentiated from 10 6 monocytes (right panel). (D) Expression levels of CD14 and DC-SIGN in control and SLAMF5 knockdown moDCs were measured by flow cytometry. Representative histograms show protein expression in control (thin line with gray shading) and knockdown cells (bolded black line) or staining with isotype control antibody (thin gray line). Bars show the relative fluorescence intensity values of CD14 and DC-SIGN, calculated using the respective isotype-matched control antibodies. The results shown are taken from four independent donors. (E) Representative histograms show CD1a expression in control (thin line with gray shading) and SLAMF5 -silenced (bolded black line) moDCs. Bars show the percentage of CD1a + cells. Data are presented as means ± SD of six independent experiments. Error bars indicate SD (* p

    Journal: Frontiers in Immunology

    Article Title: Signaling Lymphocyte Activation Molecule Family 5 Enhances Autophagy and Fine-Tunes Cytokine Response in Monocyte-Derived Dendritic Cells via Stabilization of Interferon Regulatory Factor 8

    doi: 10.3389/fimmu.2018.00062

    Figure Lengend Snippet: The effect of SLAMF5 silencing on the phenotype of moDCs. (A) Cell surface expression of SLAMF5 on monocytes and on GM-CSF/IL-4-differentiated moDCs treated or not with LPS/IFNγ was assessed by flow cytometry. (B) Monocytes were transfected with the indicated siRNAs and differentiated into moDCs. On day 5, SLAMF5 expression was measured by flow cytometry and western blot analysis. β-actin was used as protein loading control. (C) Viability of control and SLAMF5 knockdown moDCs was determined by 7-aminoactinomycin-D (7-AAD) dye exclusion. Bar graphs indicate percentage of cells negative for 7-AAD (left panel) and the total number of moDCs differentiated from 10 6 monocytes (right panel). (D) Expression levels of CD14 and DC-SIGN in control and SLAMF5 knockdown moDCs were measured by flow cytometry. Representative histograms show protein expression in control (thin line with gray shading) and knockdown cells (bolded black line) or staining with isotype control antibody (thin gray line). Bars show the relative fluorescence intensity values of CD14 and DC-SIGN, calculated using the respective isotype-matched control antibodies. The results shown are taken from four independent donors. (E) Representative histograms show CD1a expression in control (thin line with gray shading) and SLAMF5 -silenced (bolded black line) moDCs. Bars show the percentage of CD1a + cells. Data are presented as means ± SD of six independent experiments. Error bars indicate SD (* p

    Article Snippet: Flow Cytometry Viability of the cells was determined on day 5 by 7-aminoactinomycin-D (7-AAD 10 µg ml−1 ; Sigma-Aldrich) staining for 15 min immediately before flow cytometry.

    Techniques: Expressing, Flow Cytometry, Cytometry, Transfection, Western Blot, Staining, Fluorescence

    Shorter shRNA length reduced, but did not eliminate, cytotoxicity. A represents flow cytometry analysis of IS-1 cells 4 and 10 days after transduction with U6PT, sh319, sh321, sh323 and sh325 vectors. GFP expression is detected, and the percentage of GFP expressing cells was determined using the M1 gating shown (percentage GFP positive cells is shown in each histogram). B shows a comparative analysis of PAI-2 and OAS1 mRNA, in the samples described in A, 4 days after transduction. Data were generated by QRT-PCR and error bars are as described in previous figures. In C, cell lysates from samples of transduced cells described in A and B were subjected to immunoblotting with anti-PAI-2 monoclonal antibodies. Ponceau S staining served as a gel loading control, as did comparison of a single non-specific band (NS) in the immunoblot. D shows QRT-PCR analysis, as in B, for IS-1 cell mRNAs after transduction with or without U6PT, sh319, sh319scr and sh321 vectors.

    Journal: BMC Molecular Biology

    Article Title: Short-term cytotoxic effects and long-term instability of RNAi delivered using lentiviral vectors

    doi: 10.1186/1471-2199-5-9

    Figure Lengend Snippet: Shorter shRNA length reduced, but did not eliminate, cytotoxicity. A represents flow cytometry analysis of IS-1 cells 4 and 10 days after transduction with U6PT, sh319, sh321, sh323 and sh325 vectors. GFP expression is detected, and the percentage of GFP expressing cells was determined using the M1 gating shown (percentage GFP positive cells is shown in each histogram). B shows a comparative analysis of PAI-2 and OAS1 mRNA, in the samples described in A, 4 days after transduction. Data were generated by QRT-PCR and error bars are as described in previous figures. In C, cell lysates from samples of transduced cells described in A and B were subjected to immunoblotting with anti-PAI-2 monoclonal antibodies. Ponceau S staining served as a gel loading control, as did comparison of a single non-specific band (NS) in the immunoblot. D shows QRT-PCR analysis, as in B, for IS-1 cell mRNAs after transduction with or without U6PT, sh319, sh319scr and sh321 vectors.

    Article Snippet: Flow cytometry Flow cytometry was performed using Becton Dickinson FACScan, FACStrack or FACScalibur instruments at the Geneva University Medical Centre flow cytometry facility.

    Techniques: shRNA, Flow Cytometry, Cytometry, Transduction, Expressing, Generated, Quantitative RT-PCR, Staining

    PAI-2-targeted RNAi with overexpression of functional PAI-2 in IS-1 cells. IS-1 cells were transduced with a lentiviral vector for delivery of the human PAI-2 cDNA under control of the CMV promoter. A shows a flow cytometry analysis for detection of PAI-2 in transduced IS-1 PAI-2, and non-transduced IS-1 cells. Both cell types were fixed, permeabilised and labelled with anti-PAI-2 monoclonal antibodies, then incubated with PE-labelled secondary antibodies. In B, the functional activity of overexpressed PAI-2 was assessed by immunoblotting of cell lysates, for PAI-2 expression, with or without the addition of 10U of u-PA. u-PA alone is included in lane 1. In C, PAI-2 protein levels were assessed in cell lysates from IS-1 PAI-2 cells, after transduction with U6PT, sh319, sh321, sh323 and sh325 vectors. NS in B and C highlights a single non-specific band which is consistently detected in PAI-2 immunoblots of IS-1 cell lysates. D represents flow cytometry analysis of IS-1 PAI-2 cells 4 and 10 days after transduction with U6PT, sh319, sh321, sh323 and sh325 vectors. GFP expression is detected, and the percentage of GFP expressing cells was determined using the M1 gating shown (percentage GFP positive cells are given in each histogram). E and F show comparative analyses of PAI-2 and OAS1 mRNA expression, respectively, in samples described in D. Samples were analysed 4 days after transduction. Data were generated by QRT-PCR, each target gene was detected in duplicate, error bars represent the standard deviation of mean values.

    Journal: BMC Molecular Biology

    Article Title: Short-term cytotoxic effects and long-term instability of RNAi delivered using lentiviral vectors

    doi: 10.1186/1471-2199-5-9

    Figure Lengend Snippet: PAI-2-targeted RNAi with overexpression of functional PAI-2 in IS-1 cells. IS-1 cells were transduced with a lentiviral vector for delivery of the human PAI-2 cDNA under control of the CMV promoter. A shows a flow cytometry analysis for detection of PAI-2 in transduced IS-1 PAI-2, and non-transduced IS-1 cells. Both cell types were fixed, permeabilised and labelled with anti-PAI-2 monoclonal antibodies, then incubated with PE-labelled secondary antibodies. In B, the functional activity of overexpressed PAI-2 was assessed by immunoblotting of cell lysates, for PAI-2 expression, with or without the addition of 10U of u-PA. u-PA alone is included in lane 1. In C, PAI-2 protein levels were assessed in cell lysates from IS-1 PAI-2 cells, after transduction with U6PT, sh319, sh321, sh323 and sh325 vectors. NS in B and C highlights a single non-specific band which is consistently detected in PAI-2 immunoblots of IS-1 cell lysates. D represents flow cytometry analysis of IS-1 PAI-2 cells 4 and 10 days after transduction with U6PT, sh319, sh321, sh323 and sh325 vectors. GFP expression is detected, and the percentage of GFP expressing cells was determined using the M1 gating shown (percentage GFP positive cells are given in each histogram). E and F show comparative analyses of PAI-2 and OAS1 mRNA expression, respectively, in samples described in D. Samples were analysed 4 days after transduction. Data were generated by QRT-PCR, each target gene was detected in duplicate, error bars represent the standard deviation of mean values.

    Article Snippet: Flow cytometry Flow cytometry was performed using Becton Dickinson FACScan, FACStrack or FACScalibur instruments at the Geneva University Medical Centre flow cytometry facility.

    Techniques: Over Expression, Functional Assay, Transduction, Plasmid Preparation, Flow Cytometry, Cytometry, Incubation, Activity Assay, Expressing, Western Blot, Generated, Quantitative RT-PCR, Standard Deviation

    Long-term gene silencing is not stable, despite persistent marker gene expression. In A, IS-1 cells transduced with EGFP control or sh119 vectors were analysed by flow cytometry 3 and 17 days after transduction, and compared to non-transduced cells. EGFP expressing cells, from both transduced cell populations, were selected by cell sorting and named EGFPs and sh119s. 39 days after transduction, these cells were analysed for EGFP expression. Percentage EGFP positive cells, assessed by the M1 gating shown, are given in each histogram. In B, PAI-2 expression in EGFP and sh119 vector-transduced cell lysates were analysed by immunoblotting, 10 days after transduction. C is the same PAI-2 immunoblot as B, performed using EGFPs and sh119s cell lysates, 33 days after transduction.

    Journal: BMC Molecular Biology

    Article Title: Short-term cytotoxic effects and long-term instability of RNAi delivered using lentiviral vectors

    doi: 10.1186/1471-2199-5-9

    Figure Lengend Snippet: Long-term gene silencing is not stable, despite persistent marker gene expression. In A, IS-1 cells transduced with EGFP control or sh119 vectors were analysed by flow cytometry 3 and 17 days after transduction, and compared to non-transduced cells. EGFP expressing cells, from both transduced cell populations, were selected by cell sorting and named EGFPs and sh119s. 39 days after transduction, these cells were analysed for EGFP expression. Percentage EGFP positive cells, assessed by the M1 gating shown, are given in each histogram. In B, PAI-2 expression in EGFP and sh119 vector-transduced cell lysates were analysed by immunoblotting, 10 days after transduction. C is the same PAI-2 immunoblot as B, performed using EGFPs and sh119s cell lysates, 33 days after transduction.

    Article Snippet: Flow cytometry Flow cytometry was performed using Becton Dickinson FACScan, FACStrack or FACScalibur instruments at the Geneva University Medical Centre flow cytometry facility.

    Techniques: Marker, Expressing, Transduction, Flow Cytometry, Cytometry, FACS, Plasmid Preparation

    Effective gene silencing using lentiviral vectors for RNAi. The gene transfer cassette common to each vector for RNAi is shown in A. Each construction for RNAi was designed for expression of a shRNA, homologous to the target mRNA or with a scrambled sequence, driven by the RNA polymerase III-controlled human U6 promoter and ending with a terminator (T) sequence. The shRNA is represented by two arrows which encode 19 to 25 nucleotide complementary sequences and are joined by an eight nucleotide loop (L). EGFP expression is via the EF-1α promoter, oriented in the opposite direction, driving an IRES sequence and the EGFP gene. Each cassette is flanked by the HIV long terminal repeats (LTR), of which the 3' LTR is modified to ensure that the vectors are self-inactivating upon integration (SIN). B shows flow cytometry analysis of non-transduced IS-1 cells and cells four days after transduction with the U6PT control vector, a vector for expression of shRNA complementary to a region of the PAI-2 mRNA (sh325) and a vector for expression of a shRNA with a scrambled sh325 sequence (sh325scr). C shows an immunoblot for detection of PAI-2 in the cell lysate of these cells. NS highlights a single non-specific band which is consistently detected in PAI-2 immunoblots using IS-1 cell lysates. In D, PAI-2 mRNA levels from the same samples are measured by QRT-PCR of cDNA, using the ΔCT method and hypoxanthine phosphoribosyl transferase (HPRT) as the control gene. Each target gene was detected in duplicate, error bars represent the standard deviation of mean values.

    Journal: BMC Molecular Biology

    Article Title: Short-term cytotoxic effects and long-term instability of RNAi delivered using lentiviral vectors

    doi: 10.1186/1471-2199-5-9

    Figure Lengend Snippet: Effective gene silencing using lentiviral vectors for RNAi. The gene transfer cassette common to each vector for RNAi is shown in A. Each construction for RNAi was designed for expression of a shRNA, homologous to the target mRNA or with a scrambled sequence, driven by the RNA polymerase III-controlled human U6 promoter and ending with a terminator (T) sequence. The shRNA is represented by two arrows which encode 19 to 25 nucleotide complementary sequences and are joined by an eight nucleotide loop (L). EGFP expression is via the EF-1α promoter, oriented in the opposite direction, driving an IRES sequence and the EGFP gene. Each cassette is flanked by the HIV long terminal repeats (LTR), of which the 3' LTR is modified to ensure that the vectors are self-inactivating upon integration (SIN). B shows flow cytometry analysis of non-transduced IS-1 cells and cells four days after transduction with the U6PT control vector, a vector for expression of shRNA complementary to a region of the PAI-2 mRNA (sh325) and a vector for expression of a shRNA with a scrambled sh325 sequence (sh325scr). C shows an immunoblot for detection of PAI-2 in the cell lysate of these cells. NS highlights a single non-specific band which is consistently detected in PAI-2 immunoblots using IS-1 cell lysates. In D, PAI-2 mRNA levels from the same samples are measured by QRT-PCR of cDNA, using the ΔCT method and hypoxanthine phosphoribosyl transferase (HPRT) as the control gene. Each target gene was detected in duplicate, error bars represent the standard deviation of mean values.

    Article Snippet: Flow cytometry Flow cytometry was performed using Becton Dickinson FACScan, FACStrack or FACScalibur instruments at the Geneva University Medical Centre flow cytometry facility.

    Techniques: Plasmid Preparation, Expressing, shRNA, Sequencing, Modification, Flow Cytometry, Cytometry, Transduction, Western Blot, Quantitative RT-PCR, Standard Deviation