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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 1251 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 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore fluorescence activated cell sorting
    <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.
    Fluorescence Activated Cell Sorting, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 47 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 flow cytometry
    Flow <t>cytometry</t> analyses of BMSCs concentrations in peripheral blood CD34−/CD29 + and CXCR4 + are surface markers of BMSC and the graphs show the concentration of BMSCs in peripheral blood. CD29 was labeled with PE/Cy5, CD34 with PE and CXCR4 with Alexa Fluor ® 488. Specific values of CD34−/CD29 + cells and CXCR4 + cells were marked in the corresponding position of the squares. The concentration of BMSCs in peripheral blood significantly increased after transfection with SDF-1α genes. ( n = 10).
    Flow Cytometry Flow Cytometry, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 99/100, based on 1297 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore flow cytometry kit
    Flow <t>cytometry</t> analyses of BMSCs concentrations in peripheral blood CD34−/CD29 + and CXCR4 + are surface markers of BMSC and the graphs show the concentration of BMSCs in peripheral blood. CD29 was labeled with PE/Cy5, CD34 with PE and CXCR4 with Alexa Fluor ® 488. Specific values of CD34−/CD29 + cells and CXCR4 + cells were marked in the corresponding position of the squares. The concentration of BMSCs in peripheral blood significantly increased after transfection with SDF-1α genes. ( n = 10).
    Flow Cytometry Kit, supplied by Millipore, 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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    Millipore flow cytometry instrument
    Flow <t>cytometry</t> analyses of BMSCs concentrations in peripheral blood CD34−/CD29 + and CXCR4 + are surface markers of BMSC and the graphs show the concentration of BMSCs in peripheral blood. CD29 was labeled with PE/Cy5, CD34 with PE and CXCR4 with Alexa Fluor ® 488. Specific values of CD34−/CD29 + cells and CXCR4 + cells were marked in the corresponding position of the squares. The concentration of BMSCs in peripheral blood significantly increased after transfection with SDF-1α genes. ( n = 10).
    Flow Cytometry Instrument, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beckman Coulter flow cytometry assay flow cytometry
    Flow <t>cytometry</t> detection of apoptosis. dsRNA induced HepG2.2.15 cell apoptosis ( ∗ P
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    Becton Dickinson flow cytometry calibur flow cytometry
    Flow <t>cytometry</t> detection of apoptosis. dsRNA induced HepG2.2.15 cell apoptosis ( ∗ P
    Flow Cytometry Calibur Flow Cytometry, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Miltenyi Biotec flow cytometry flow cytometry
    Flow <t>cytometry</t> detection of apoptosis. dsRNA induced HepG2.2.15 cell apoptosis ( ∗ P
    Flow Cytometry Flow Cytometry, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 99/100, based on 29 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

    Construction of green fluorescence protein (GFP) insertion in sigE . (A) Along with pHCas9, pBE-gfp was used to deliver sgRNA and the donor DNA template to generate GFP insertion in the sigE gene through HDR. (B) Insertion of P xyl -gfp was confirmed with PCR and (C) Guava InCyte flow cytometry assay; 1 indicates the B. subtilis 168 control; 2 is pBE-gfp-transferred BS-C111.

    Journal: Frontiers in Microbiology

    Article Title: A Highly Efficient CRISPR-Cas9-Mediated Large Genomic Deletion in Bacillus subtilis

    doi: 10.3389/fmicb.2017.01167

    Figure Lengend Snippet: Construction of green fluorescence protein (GFP) insertion in sigE . (A) Along with pHCas9, pBE-gfp was used to deliver sgRNA and the donor DNA template to generate GFP insertion in the sigE gene through HDR. (B) Insertion of P xyl -gfp was confirmed with PCR and (C) Guava InCyte flow cytometry assay; 1 indicates the B. subtilis 168 control; 2 is pBE-gfp-transferred BS-C111.

    Article Snippet: Flow Cytometry Flow cytometry and data analysis were conducted using a Guava® easyCyte cytometer (Millipore Corporation, Billerica, MA, United States).

    Techniques: Fluorescence, Polymerase Chain Reaction, Flow Cytometry, Cytometry

    Inducible genetic ablation of MASTL using CRISPR/Cas9 techniques in breast cancer cells a Schematic representation of the sgRNA/cas9- inducible lentiviral vector used in this study. b Representative flow cytometry plots of dsRed induction in MDA-MB-231 cells expressing both Cas9 and sgRNAs against MASTL (isg MASTL ) or control cells expressing Cas9 alone (iCas9). Numbers indicate the percentage of DsRed-positive cells after 4 days treatment with doxycycline. Immunobloting analysis of MASTL protein levels in the absence or presence of doxycycline (4 days treatment) in the two MDA-MB-231 selected clones is shown on the right panel. Cas9 induction was monitored with an anti-Flag antibody. c-d Genomic analysis of isg MASTL clones. sgRNA and PAM sequence are highlighted in yellow and blue colors, respectively. Red arrowhead denotes predicted Cas9 cutting site. Note that this clone is already heterozygous for MASTL before treatment due to a 15 bp deletion that leads to a frameshift mutation and a premature stop codon c . Treatment with doxycycline results in deletion of MASTL in both alleles due to 1–43 bp deletions also causing frameshift mutations as indicated in panel d. Representative sequences are shown

    Journal: Cell Death and Differentiation

    Article Title: Therapeutic relevance of the PP2A-B55 inhibitory kinase MASTL/Greatwall in breast cancer

    doi: 10.1038/s41418-017-0024-0

    Figure Lengend Snippet: Inducible genetic ablation of MASTL using CRISPR/Cas9 techniques in breast cancer cells a Schematic representation of the sgRNA/cas9- inducible lentiviral vector used in this study. b Representative flow cytometry plots of dsRed induction in MDA-MB-231 cells expressing both Cas9 and sgRNAs against MASTL (isg MASTL ) or control cells expressing Cas9 alone (iCas9). Numbers indicate the percentage of DsRed-positive cells after 4 days treatment with doxycycline. Immunobloting analysis of MASTL protein levels in the absence or presence of doxycycline (4 days treatment) in the two MDA-MB-231 selected clones is shown on the right panel. Cas9 induction was monitored with an anti-Flag antibody. c-d Genomic analysis of isg MASTL clones. sgRNA and PAM sequence are highlighted in yellow and blue colors, respectively. Red arrowhead denotes predicted Cas9 cutting site. Note that this clone is already heterozygous for MASTL before treatment due to a 15 bp deletion that leads to a frameshift mutation and a premature stop codon c . Treatment with doxycycline results in deletion of MASTL in both alleles due to 1–43 bp deletions also causing frameshift mutations as indicated in panel d. Representative sequences are shown

    Article Snippet: Flow-cytometry Flow cytometry analysis of DNA content was performed by cell fixation with cold 70% Ethanol followed by staining with 10 µg/ml Propidium Iodide (Sigma) or 10 μg/ml Hoechst 3342 (Molecular Probes, Thermofisher).

    Techniques: CRISPR, Plasmid Preparation, Flow Cytometry, Cytometry, Multiple Displacement Amplification, Expressing, Western Blot, Clone Assay, Sequencing, Mutagenesis

    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

    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

    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

    Growth of Streptococcus thermophilus DSM 20617 T (squares) and its derivative A33 (circles) strain in M17 broth in the presence of sucrose, and lactose. (A) Growth of S. thermophilus strains in the presence of sucrose at a final concentration of 0.2% (white symbols) and 1% (black symbols). Strain DSM 20617 T (autolytic) (white and black squares), strain A33 (non-autolytic) (white and black circles). Flow cytometry of SYBR Green I stained cells of S. thermophilus DSM 20617 T grown in M17 (0.2 % wt/v sucrose). (B) Cells after 4 h of growth at 37°C (blue gate). (C) Cells and cell debris (red gate) after 8 h of growth at 37°C. (D) Cells and cell debris (red gate) after 8 h of growth at 37°C treated with DNAse. (E) M17 broth SYBR Green I stained.

    Journal: Frontiers in Microbiology

    Article Title: Role of Temperate Bacteriophage ϕ20617 on Streptococcus thermophilus DSM 20617T Autolysis and Biology

    doi: 10.3389/fmicb.2018.02719

    Figure Lengend Snippet: Growth of Streptococcus thermophilus DSM 20617 T (squares) and its derivative A33 (circles) strain in M17 broth in the presence of sucrose, and lactose. (A) Growth of S. thermophilus strains in the presence of sucrose at a final concentration of 0.2% (white symbols) and 1% (black symbols). Strain DSM 20617 T (autolytic) (white and black squares), strain A33 (non-autolytic) (white and black circles). Flow cytometry of SYBR Green I stained cells of S. thermophilus DSM 20617 T grown in M17 (0.2 % wt/v sucrose). (B) Cells after 4 h of growth at 37°C (blue gate). (C) Cells and cell debris (red gate) after 8 h of growth at 37°C. (D) Cells and cell debris (red gate) after 8 h of growth at 37°C treated with DNAse. (E) M17 broth SYBR Green I stained.

    Article Snippet: Flow Cytometry Measurements The cell suspensions were diluted in sterile filtered (0.2 μm) water and stained with a cell-permeant SYBR green I (1X) (Sigma-Aldrich, Milan, Italy) or to a double staining using SYBR green I and propidium iodide (PI) ( ).

    Techniques: Concentration Assay, Flow Cytometry, Cytometry, SYBR Green Assay, 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

    Blocking IL-1R relieves disease symptoms. iOvaFla; LysM-Cre +/− mice with significant joint swelling at 4.0 mm were administered either anti-IL-1R blocking antibody or isotype control every 3–4 days for 2 weeks. a Their weights and tibiotarsal joints were measured during that time ( n = 7 biological replicates per treatment). b Post-treatment serum cytokines measured by a BD Biosciences cytokine bead array and IL-18 ELISA. c Flow cytometry and d quantification of monocytes (MO; CD11b + Ly6C Hi Ly6G Lo ) and neutrophils (NE; CD11b + Ly6C Lo Ly6G Hi ). For each flow cytometry plot, the top row is from the spleen and the second row from peripheral and mesenteric lymph nodes. Each column represents a different treatment. Data in b – d are representative of three independent experiments. Error bars are s.d. Results were analysed with either a Mann–Whitney test or two-way ANOVA and Bonferroni post-tests; * p

    Journal: Nature Communications

    Article Title: NAIP/NLRC4 inflammasome activation in MRP8+ cells is sufficient to cause systemic inflammatory disease

    doi: 10.1038/s41467-017-02266-w

    Figure Lengend Snippet: Blocking IL-1R relieves disease symptoms. iOvaFla; LysM-Cre +/− mice with significant joint swelling at 4.0 mm were administered either anti-IL-1R blocking antibody or isotype control every 3–4 days for 2 weeks. a Their weights and tibiotarsal joints were measured during that time ( n = 7 biological replicates per treatment). b Post-treatment serum cytokines measured by a BD Biosciences cytokine bead array and IL-18 ELISA. c Flow cytometry and d quantification of monocytes (MO; CD11b + Ly6C Hi Ly6G Lo ) and neutrophils (NE; CD11b + Ly6C Lo Ly6G Hi ). For each flow cytometry plot, the top row is from the spleen and the second row from peripheral and mesenteric lymph nodes. Each column represents a different treatment. Data in b – d are representative of three independent experiments. Error bars are s.d. Results were analysed with either a Mann–Whitney test or two-way ANOVA and Bonferroni post-tests; * p

    Article Snippet: Flow cytometry Spleens and lymph nodes were collected from euthanized mice, minced with scissors, and incubated in RPMI with 5% FBS containing collagenase VIII (1 mg/mL, Sigma) at 37 °C for 45 min. After straining the digested spleens and lymph nodes, single-cell suspensions were treated with ACK Lysing Buffer (Invitrogen A1049201) to lyse erythrocytes.

    Techniques: Blocking Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, MANN-WHITNEY

    Neutrophil iOvaFla expression is sufficient for inflammatory disease. a Weight and tibiotarsal joint swelling as iOvaFla; MRP8-Cre −/− and iOvaFla; MRP8-Cre +/− mice age ( n = 6 biological replicates per genotype). b Serum cytokines measured by a BD Biosciences cytokine bead array and IL-18 ELISA of 10–12-week-old mice. c Flow cytometry and d quantification of monocytes (MO; CD11b + Ly6C Hi Ly6G Lo ) and neutrophils (NE; CD11b + Ly6C Lo Ly6G Hi ). For each flow cytometry plot, the top row is from the spleen and the second row from peripheral and mesenteric lymph nodes. Each column represents a different genotype. e Weight and tibiotarsal joint measurements after OVA-Fla; MRP8-Cre +/− mice were administered either anti-IL-1R blocking antibody or isotype control every 3–4 days for 2 weeks ( n = 3 biological replicates per treatment). Data in b – d are representative of three independent experiments. Error bars are s.d. Results were analysed with either a Mann–Whitney test or two-way ANOVA and Bonferroni post-tests; * p

    Journal: Nature Communications

    Article Title: NAIP/NLRC4 inflammasome activation in MRP8+ cells is sufficient to cause systemic inflammatory disease

    doi: 10.1038/s41467-017-02266-w

    Figure Lengend Snippet: Neutrophil iOvaFla expression is sufficient for inflammatory disease. a Weight and tibiotarsal joint swelling as iOvaFla; MRP8-Cre −/− and iOvaFla; MRP8-Cre +/− mice age ( n = 6 biological replicates per genotype). b Serum cytokines measured by a BD Biosciences cytokine bead array and IL-18 ELISA of 10–12-week-old mice. c Flow cytometry and d quantification of monocytes (MO; CD11b + Ly6C Hi Ly6G Lo ) and neutrophils (NE; CD11b + Ly6C Lo Ly6G Hi ). For each flow cytometry plot, the top row is from the spleen and the second row from peripheral and mesenteric lymph nodes. Each column represents a different genotype. e Weight and tibiotarsal joint measurements after OVA-Fla; MRP8-Cre +/− mice were administered either anti-IL-1R blocking antibody or isotype control every 3–4 days for 2 weeks ( n = 3 biological replicates per treatment). Data in b – d are representative of three independent experiments. Error bars are s.d. Results were analysed with either a Mann–Whitney test or two-way ANOVA and Bonferroni post-tests; * p

    Article Snippet: Flow cytometry Spleens and lymph nodes were collected from euthanized mice, minced with scissors, and incubated in RPMI with 5% FBS containing collagenase VIII (1 mg/mL, Sigma) at 37 °C for 45 min. After straining the digested spleens and lymph nodes, single-cell suspensions were treated with ACK Lysing Buffer (Invitrogen A1049201) to lyse erythrocytes.

    Techniques: Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Blocking Assay, MANN-WHITNEY

    Genetic system for inducible NLRC4 inflammasome activation in vivo. a Schematic showing iOvaFla transgene insertion in the Rosa26 locus. b Flow cytometry analysis of bone marrow derived macrophages (BMMs) cultured from WT, Nlrc4 −/− ; iOvaFla and Nlrc4 −/− ; iOvaFla; LysM-Cre +/− mice (representative images from three biological replicates), c peritoneal macrophages (CD11b + F4/80 + ) from iOvaFla; LysM-Cre −/− , iOvaFla; LysM-Cre +/− , and Nlrc4 −/− ; iOvaFla; LysM-Cre +/− mice, and d neutrophils (CD11b + Ly6G Hi , Ly6C Lo ) from the bone marrow, spleens and lymph nodes. Data in b – d are representative of two independent experiments. Error bars are s.d. Results were analysed with a two-way ANOVA and Bonferroni post-tests; * p

    Journal: Nature Communications

    Article Title: NAIP/NLRC4 inflammasome activation in MRP8+ cells is sufficient to cause systemic inflammatory disease

    doi: 10.1038/s41467-017-02266-w

    Figure Lengend Snippet: Genetic system for inducible NLRC4 inflammasome activation in vivo. a Schematic showing iOvaFla transgene insertion in the Rosa26 locus. b Flow cytometry analysis of bone marrow derived macrophages (BMMs) cultured from WT, Nlrc4 −/− ; iOvaFla and Nlrc4 −/− ; iOvaFla; LysM-Cre +/− mice (representative images from three biological replicates), c peritoneal macrophages (CD11b + F4/80 + ) from iOvaFla; LysM-Cre −/− , iOvaFla; LysM-Cre +/− , and Nlrc4 −/− ; iOvaFla; LysM-Cre +/− mice, and d neutrophils (CD11b + Ly6G Hi , Ly6C Lo ) from the bone marrow, spleens and lymph nodes. Data in b – d are representative of two independent experiments. Error bars are s.d. Results were analysed with a two-way ANOVA and Bonferroni post-tests; * p

    Article Snippet: Flow cytometry Spleens and lymph nodes were collected from euthanized mice, minced with scissors, and incubated in RPMI with 5% FBS containing collagenase VIII (1 mg/mL, Sigma) at 37 °C for 45 min. After straining the digested spleens and lymph nodes, single-cell suspensions were treated with ACK Lysing Buffer (Invitrogen A1049201) to lyse erythrocytes.

    Techniques: Activation Assay, In Vivo, Flow Cytometry, Cytometry, Derivative Assay, Cell Culture, Mouse Assay

    Inflammasome-driven disease leads to myeloid cell hyperplasia. a Haematocrit concentration and cell numbers from a complete blood count (CBC) performed on the blood of 10-week-old iOvaFla; LysM-Cre +/− and iOvaFla; LysM-Cre –/− mice. b Flow cytometry and c quantification of total monocytes (MO; CD11b + Ly6C Hi Ly6G Lo ) and neutrophils (NE; CD11b + Ly6C Lo Ly6G Hi ) from mice aged 10–16 weeks old. Flow cytometry plots are representative of three separate experiments. Dot plot percentages are from CD11b + gate, and cell numbers are calculated from percentage of live cells (total cells). Data in a are representative of two independent experiments; Data in b , c are representative of three independent experiments. Error bars are s.d. Results were analysed with either a Mann–Whitney test or two-way ANOVA and Bonferroni post-tests; * p

    Journal: Nature Communications

    Article Title: NAIP/NLRC4 inflammasome activation in MRP8+ cells is sufficient to cause systemic inflammatory disease

    doi: 10.1038/s41467-017-02266-w

    Figure Lengend Snippet: Inflammasome-driven disease leads to myeloid cell hyperplasia. a Haematocrit concentration and cell numbers from a complete blood count (CBC) performed on the blood of 10-week-old iOvaFla; LysM-Cre +/− and iOvaFla; LysM-Cre –/− mice. b Flow cytometry and c quantification of total monocytes (MO; CD11b + Ly6C Hi Ly6G Lo ) and neutrophils (NE; CD11b + Ly6C Lo Ly6G Hi ) from mice aged 10–16 weeks old. Flow cytometry plots are representative of three separate experiments. Dot plot percentages are from CD11b + gate, and cell numbers are calculated from percentage of live cells (total cells). Data in a are representative of two independent experiments; Data in b , c are representative of three independent experiments. Error bars are s.d. Results were analysed with either a Mann–Whitney test or two-way ANOVA and Bonferroni post-tests; * p

    Article Snippet: Flow cytometry Spleens and lymph nodes were collected from euthanized mice, minced with scissors, and incubated in RPMI with 5% FBS containing collagenase VIII (1 mg/mL, Sigma) at 37 °C for 45 min. After straining the digested spleens and lymph nodes, single-cell suspensions were treated with ACK Lysing Buffer (Invitrogen A1049201) to lyse erythrocytes.

    Techniques: Concentration Assay, Mouse Assay, Flow Cytometry, Cytometry, MANN-WHITNEY

    Effects of LUM on apoptosis of Nalm-6 cells. Flow cytometry analysis results demonstrated that the proportion of total apoptotic cells decreased in the Nalm-6 cells of the BM-MSCs-LUM-329 and 437 groups compared with Nalm-6+BM-MSCs group. Compared with Nalm-6+BM-MSCs group, total apoptotic cells remained unchanged in BM-MSCs-cDNA3.1-LUM group. *P

    Journal: Oncology Letters

    Article Title: Leukemia stem cells promote chemoresistance by inducing downregulation of lumican in mesenchymal stem cells

    doi: 10.3892/ol.2019.10767

    Figure Lengend Snippet: Effects of LUM on apoptosis of Nalm-6 cells. Flow cytometry analysis results demonstrated that the proportion of total apoptotic cells decreased in the Nalm-6 cells of the BM-MSCs-LUM-329 and 437 groups compared with Nalm-6+BM-MSCs group. Compared with Nalm-6+BM-MSCs group, total apoptotic cells remained unchanged in BM-MSCs-cDNA3.1-LUM group. *P

    Article Snippet: Cells were analyzed on a flow cytometry system (Guava easyCyte8HT; EMD Millipore) with the Guava Incyte software (version 2.8; EMD Millipore).

    Techniques: Flow Cytometry, Cytometry

    T-cell intrinsic TLR2 signals are not required for effector T-cell or Treg functions in vivo . (A) CD4 + T cells from the spleen, MLN and colonic LPL were analyzed for Foxp3 expression by flow cytometry. Data represents group means (±SEM) from two pooled independent experiments ( n =8–10 mice per group). (B) NaYve CD45RB hi (RB hi ), memory CD25 − CD45RB lo (CD25 − ) and Treg CD25 + CD45RB lo (CD25 + ) CD4 + T cells were sorted from the MLN and LPL of B6 WT mice ( n =5) and analyzed for TLR2 gene expression by Q-PCR. Data represent means±SEM normalized relative to HPRT. (C) B6.RAG −/− mice were reconstituted with either 4×10 5 WT or TLR2 −/− CD4 + CD45RB hi T cells alone (colitis “induction”) or concurrently with 2×10 5 WT or TLR2 −/− CD4 + CD25 + Treg (colitis “protection”). After 8–10 wk colonic pathology was assessed histologically. Each symbol represents a single animal and the data represents pooled results of two independent experiments ( n =10–14 mice per group). Horizontal lines represent group means and differences were assessed using the Mann–Whitney U -test; ns, not significant.

    Journal: European Journal of Immunology

    Article Title: TLR2-independent induction and regulation of chronic intestinal inflammation

    doi: 10.1002/eji.200939669

    Figure Lengend Snippet: T-cell intrinsic TLR2 signals are not required for effector T-cell or Treg functions in vivo . (A) CD4 + T cells from the spleen, MLN and colonic LPL were analyzed for Foxp3 expression by flow cytometry. Data represents group means (±SEM) from two pooled independent experiments ( n =8–10 mice per group). (B) NaYve CD45RB hi (RB hi ), memory CD25 − CD45RB lo (CD25 − ) and Treg CD25 + CD45RB lo (CD25 + ) CD4 + T cells were sorted from the MLN and LPL of B6 WT mice ( n =5) and analyzed for TLR2 gene expression by Q-PCR. Data represent means±SEM normalized relative to HPRT. (C) B6.RAG −/− mice were reconstituted with either 4×10 5 WT or TLR2 −/− CD4 + CD45RB hi T cells alone (colitis “induction”) or concurrently with 2×10 5 WT or TLR2 −/− CD4 + CD25 + Treg (colitis “protection”). After 8–10 wk colonic pathology was assessed histologically. Each symbol represents a single animal and the data represents pooled results of two independent experiments ( n =10–14 mice per group). Horizontal lines represent group means and differences were assessed using the Mann–Whitney U -test; ns, not significant.

    Article Snippet: Flow cytometry Aliquots of ∼1×106 spleen cells in HBSS/0.1% BSA (Sigma Aldrich) were incubated for 30 min with FcR block (eBioscience) at 4°C, and surface staining steps performed for 20 min at 4°C with a panel of monoclonal antibodies as follows: biotinylated anti-DX5, biotinylated anti-Gr1, PE anti-CD11c and APC anti-CD11b (all obtained from BD Bioscience).

    Techniques: In Vivo, Expressing, Flow Cytometry, Cytometry, Mouse Assay, Polymerase Chain Reaction, MANN-WHITNEY

    EIF5A2 overexpression reversed the effects of miR-494-3p knockdown on proliferation, invasion, and apoptosis of medulloblastoma cells. ( A ) RT-PCR and IHC revealed the expression level of EIF5A2 in 42 medulloblastoma samples. ( B ) Western blot showed the expression level of EIF5A2 in Daoy and D341 cells after transfected with miR-494-3p or miR-494-3p+EIF5A2. ( C ) The CCK-8 assay was used to detect cell viability of Daoy and D341 cell transfected with miR-494-3p or miR-494-3p+EIF5A2 at the indicated time points. ( D ) Flow cytometry was used to evaluate apoptosis of Daoy and D341 cell transfected with miR-494-3p or miR-494-3p+EIF5A2. ( E, F ) Transwell assay showed the invaded cell number in Daoy and D341 cells transfected with miR-494-3p or miR-494-3p + EIF5A2. ( G ) The expression of E-cadherin, N-cadherin and Vimentin proteins in Daoy and D341 cells transfected with miR-494-3p or miR-494-3p + EIF5A2 were detected by Western blot.* P

    Journal: OncoTargets and therapy

    Article Title: Long Noncoding RNA TP73-AS1 Modulates Medulloblastoma Progression In Vitro And In Vivo By Sponging miR-494-3p And Targeting EIF5A2

    doi: 10.2147/OTT.S228305

    Figure Lengend Snippet: EIF5A2 overexpression reversed the effects of miR-494-3p knockdown on proliferation, invasion, and apoptosis of medulloblastoma cells. ( A ) RT-PCR and IHC revealed the expression level of EIF5A2 in 42 medulloblastoma samples. ( B ) Western blot showed the expression level of EIF5A2 in Daoy and D341 cells after transfected with miR-494-3p or miR-494-3p+EIF5A2. ( C ) The CCK-8 assay was used to detect cell viability of Daoy and D341 cell transfected with miR-494-3p or miR-494-3p+EIF5A2 at the indicated time points. ( D ) Flow cytometry was used to evaluate apoptosis of Daoy and D341 cell transfected with miR-494-3p or miR-494-3p+EIF5A2. ( E, F ) Transwell assay showed the invaded cell number in Daoy and D341 cells transfected with miR-494-3p or miR-494-3p + EIF5A2. ( G ) The expression of E-cadherin, N-cadherin and Vimentin proteins in Daoy and D341 cells transfected with miR-494-3p or miR-494-3p + EIF5A2 were detected by Western blot.* P

    Article Snippet: Flow Cytometry Assay The PI/Annexin V Cell Apoptosis Kit (Sigma) was used to detect cell apoptosis, Daoy and D341 cells were gathered and washed with PBS for 3 times, approximately 1 × 106 cells were resuspended in 200 μL binding buffer, then PI and FITC Annexin V were added to Daoy and D341 cells, and incubated for 15 min at 37°C in the dark, then the cell apoptosis was analyzed by flow cytometry.

    Techniques: Over Expression, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry, Expressing, Western Blot, Transfection, CCK-8 Assay, Flow Cytometry, Cytometry, Transwell Assay

    The knockdown of LncRNA TP73-AS1 inhibited cell proliferation, migration, invasion, and induced apoptosis of medulloblastoma cells. ( A ) RT-PCR and ISH revealed the level of TP73-AS1 in 42 medulloblastoma samples. ( B ) RT-PCR showed the expression level of TP73-AS1 after si-TP73-AS1 transfection in Daoy and D341 cells. ( C ) The proliferation ability of Daoy and D341 cells transfected with si-TP73-AS1 was detected by CCK-8. ( D ) The apoptosis ability of Daoy and D341 cells transfected with si-TP73-AS1 was detected by flow cytometry. ( E, F ) transwell assay showed the number of invading cells after si-TP73-AS1 was transfected into Daoy and D341 cells. ( G ) The expression of E-cadherin, N-cadherin and Vimentin proteins in Daoy and D341 cells transfected with si-TP73-AS1 was detected by Western blot.* P

    Journal: OncoTargets and therapy

    Article Title: Long Noncoding RNA TP73-AS1 Modulates Medulloblastoma Progression In Vitro And In Vivo By Sponging miR-494-3p And Targeting EIF5A2

    doi: 10.2147/OTT.S228305

    Figure Lengend Snippet: The knockdown of LncRNA TP73-AS1 inhibited cell proliferation, migration, invasion, and induced apoptosis of medulloblastoma cells. ( A ) RT-PCR and ISH revealed the level of TP73-AS1 in 42 medulloblastoma samples. ( B ) RT-PCR showed the expression level of TP73-AS1 after si-TP73-AS1 transfection in Daoy and D341 cells. ( C ) The proliferation ability of Daoy and D341 cells transfected with si-TP73-AS1 was detected by CCK-8. ( D ) The apoptosis ability of Daoy and D341 cells transfected with si-TP73-AS1 was detected by flow cytometry. ( E, F ) transwell assay showed the number of invading cells after si-TP73-AS1 was transfected into Daoy and D341 cells. ( G ) The expression of E-cadherin, N-cadherin and Vimentin proteins in Daoy and D341 cells transfected with si-TP73-AS1 was detected by Western blot.* P

    Article Snippet: Flow Cytometry Assay The PI/Annexin V Cell Apoptosis Kit (Sigma) was used to detect cell apoptosis, Daoy and D341 cells were gathered and washed with PBS for 3 times, approximately 1 × 106 cells were resuspended in 200 μL binding buffer, then PI and FITC Annexin V were added to Daoy and D341 cells, and incubated for 15 min at 37°C in the dark, then the cell apoptosis was analyzed by flow cytometry.

    Techniques: Migration, Reverse Transcription Polymerase Chain Reaction, In Situ Hybridization, Expressing, Transfection, CCK-8 Assay, Flow Cytometry, Cytometry, Transwell Assay, Western Blot

    Inhibition of miR-494-3p expression reversed the effects of TP73-AS1 knockdown on proliferation, invasion, and apoptosis of medulloblastoma cells. ( A ) RT-PCR and ISH revealed the expression level of miR-494-3p in 42 medulloblastoma samples. ( B ) RT-PCR showed the expression level of TP73-AS1 after transfected with si-TP73-AS1 or si-TP73-AS1+anti-miR-494-3p in Daoy and D341 cells. ( C ) The proliferation ability of Daoy and D341 cells transfected with si-TP73-AS1 or si-TP73-AS1+ anti-miR-494-3p were detected by CCK-8. ( D ) The apoptosis ability of Daoy and D341 cells transfected with si-TP73-AS1 or si-TP73-AS1+anti-miR-494-3p were detected by flow cytometry. ( E, F ) Transwell assay showed the number of invading cells after si-TP73-AS1 or si-TP73-AS1+anti-miR-494-3p were transfected into Daoy and D341 cells. ( G ) The expression of E-cadherin, N-cadherin and Vimentin proteins in Daoy and D341 cells transfected with si-TP73-AS1 or si-TP73-AS1+anti-miR-494-3p were detected by Western blot. * P

    Journal: OncoTargets and therapy

    Article Title: Long Noncoding RNA TP73-AS1 Modulates Medulloblastoma Progression In Vitro And In Vivo By Sponging miR-494-3p And Targeting EIF5A2

    doi: 10.2147/OTT.S228305

    Figure Lengend Snippet: Inhibition of miR-494-3p expression reversed the effects of TP73-AS1 knockdown on proliferation, invasion, and apoptosis of medulloblastoma cells. ( A ) RT-PCR and ISH revealed the expression level of miR-494-3p in 42 medulloblastoma samples. ( B ) RT-PCR showed the expression level of TP73-AS1 after transfected with si-TP73-AS1 or si-TP73-AS1+anti-miR-494-3p in Daoy and D341 cells. ( C ) The proliferation ability of Daoy and D341 cells transfected with si-TP73-AS1 or si-TP73-AS1+ anti-miR-494-3p were detected by CCK-8. ( D ) The apoptosis ability of Daoy and D341 cells transfected with si-TP73-AS1 or si-TP73-AS1+anti-miR-494-3p were detected by flow cytometry. ( E, F ) Transwell assay showed the number of invading cells after si-TP73-AS1 or si-TP73-AS1+anti-miR-494-3p were transfected into Daoy and D341 cells. ( G ) The expression of E-cadherin, N-cadherin and Vimentin proteins in Daoy and D341 cells transfected with si-TP73-AS1 or si-TP73-AS1+anti-miR-494-3p were detected by Western blot. * P

    Article Snippet: Flow Cytometry Assay The PI/Annexin V Cell Apoptosis Kit (Sigma) was used to detect cell apoptosis, Daoy and D341 cells were gathered and washed with PBS for 3 times, approximately 1 × 106 cells were resuspended in 200 μL binding buffer, then PI and FITC Annexin V were added to Daoy and D341 cells, and incubated for 15 min at 37°C in the dark, then the cell apoptosis was analyzed by flow cytometry.

    Techniques: Inhibition, Expressing, Reverse Transcription Polymerase Chain Reaction, In Situ Hybridization, Transfection, CCK-8 Assay, Flow Cytometry, Cytometry, Transwell Assay, Western Blot

    Increased Reactive Oxygen Is Detected Only in Oligodendrocytes (A) DCF-DA fluorescence intensity measured by flow cytometry in dissociated forebrain + optic nerves of WT and PLP;Nf1fl+ mice 6 months after tamoxifen injection. Gates for DCF-DA with cell percentages indicated. (B) Gates were used to discriminate cell types. Top row: WT animals are shown with red gates. Middle row: PLP;Nf1fl+ animals are shown with blue gates. Bottom row: DCF-DA fluorescence intensities per cell type with WT (red) and PLP;Nf1fl+ (blue) lines. (C) Cell Rox Orange fluorescence in GFP− and GFP+ cells.

    Journal: Cell reports

    Article Title: Nf1 Loss and Ras Hyperactivation in Oligodendrocytes Induce NOS-Driven Defects in Myelin and Vasculature

    doi: 10.1016/j.celrep.2013.08.011

    Figure Lengend Snippet: Increased Reactive Oxygen Is Detected Only in Oligodendrocytes (A) DCF-DA fluorescence intensity measured by flow cytometry in dissociated forebrain + optic nerves of WT and PLP;Nf1fl+ mice 6 months after tamoxifen injection. Gates for DCF-DA with cell percentages indicated. (B) Gates were used to discriminate cell types. Top row: WT animals are shown with red gates. Middle row: PLP;Nf1fl+ animals are shown with blue gates. Bottom row: DCF-DA fluorescence intensities per cell type with WT (red) and PLP;Nf1fl+ (blue) lines. (C) Cell Rox Orange fluorescence in GFP− and GFP+ cells.

    Article Snippet: Flow Cytometry Brains were perfused with 30 ml PBS and then dissected, roughly chopped, and incubated in 1 ml Accutase (Millipore) for 30 min at 37°C.

    Techniques: Fluorescence, Flow Cytometry, Cytometry, Plasmid Purification, Mouse Assay, Injection

    Flow cytometry strategy to assess the frequency and signaling of MDM-CD4+ T cell conjugates. ( A ) Schematic diagram describing the experimental protocol developed; MDMs labeled with the lipophilic membrane dye PKH26 were exposed or not to sAg (1 µg/ml SEE), before incubation with Fluo-2 leakres loaded CD4 + T cells pre-labeled with a distinct lipophilic membrane dye (DiD); DAPI stained samples were exposed or not to ionomycin and analyzed by flow cytometry ( > 10,000 events acquired per sample). ( B ) Gating strategy and output metrics. Pulse Width gated Fluo-2 leakres Mean Fluorescence Intensity signal (MFI; green bold line) was expressed as a percentage of the signal within the same gate after adding ionomycin to the sample (green dotted line). ( C ) Graphs showing cell conjugate formation over time and the intensity of calcium signals within these conjugates, for T cells pre-coated with an anti-CD3 antibody (OKT3, 5 µg/ml; Anti-CD3/Fc receptor panel), presented to SEE pre-pulsed MDMs (1 µg/ml; sAg panel) or to untreated MDMs (black lines). Each graph represents the mean (±SD) from experiments performed on cells from N = 3 donors.

    Journal: Scientific Reports

    Article Title: A correlative and quantitative imaging approach enabling characterization of primary cell-cell communication: Case of human CD4+ T cell-macrophage immunological synapses

    doi: 10.1038/s41598-018-26172-3

    Figure Lengend Snippet: Flow cytometry strategy to assess the frequency and signaling of MDM-CD4+ T cell conjugates. ( A ) Schematic diagram describing the experimental protocol developed; MDMs labeled with the lipophilic membrane dye PKH26 were exposed or not to sAg (1 µg/ml SEE), before incubation with Fluo-2 leakres loaded CD4 + T cells pre-labeled with a distinct lipophilic membrane dye (DiD); DAPI stained samples were exposed or not to ionomycin and analyzed by flow cytometry ( > 10,000 events acquired per sample). ( B ) Gating strategy and output metrics. Pulse Width gated Fluo-2 leakres Mean Fluorescence Intensity signal (MFI; green bold line) was expressed as a percentage of the signal within the same gate after adding ionomycin to the sample (green dotted line). ( C ) Graphs showing cell conjugate formation over time and the intensity of calcium signals within these conjugates, for T cells pre-coated with an anti-CD3 antibody (OKT3, 5 µg/ml; Anti-CD3/Fc receptor panel), presented to SEE pre-pulsed MDMs (1 µg/ml; sAg panel) or to untreated MDMs (black lines). Each graph represents the mean (±SD) from experiments performed on cells from N = 3 donors.

    Article Snippet: Flow cytometry strategy for assessing conjugate formation MDMs (day 9) were detached then labelled with 2 µM PKH-26 (Sigma-Aldrich) for 5 min at room temperature (RT).

    Techniques: Flow Cytometry, Cytometry, Labeling, Incubation, Staining, Fluorescence

    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

    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

    Montelukast treatment does not affect T cell proliferation Leukocytes in peripheral blood were isolated from CIA animals treated with montelukast (30 mg/kg) or vehicle control at weeks 9 post booster immunization and analyzed with flow cytometry. A. - D. Surface staining of CD45 + cells, CD4 + T cells, CD8 + T cells and B cells; E. - H. Th1, Th17 and Treg cells were analyzed by intracellular staining of IFN-γ, IL-17 and Foxp-3 respectively within the CD4 + gate. Data represents mean ± SEM of three independent experiments.

    Journal: Oncotarget

    Article Title: CysLT1 receptor antagonist alleviates pathogenesis of collagen-induced arthritis mouse model

    doi: 10.18632/oncotarget.22664

    Figure Lengend Snippet: Montelukast treatment does not affect T cell proliferation Leukocytes in peripheral blood were isolated from CIA animals treated with montelukast (30 mg/kg) or vehicle control at weeks 9 post booster immunization and analyzed with flow cytometry. A. - D. Surface staining of CD45 + cells, CD4 + T cells, CD8 + T cells and B cells; E. - H. Th1, Th17 and Treg cells were analyzed by intracellular staining of IFN-γ, IL-17 and Foxp-3 respectively within the CD4 + gate. Data represents mean ± SEM of three independent experiments.

    Article Snippet: Flow cytometry Splenocytes or leukocytes in peripheral blood were incubated for 5 hr at 37°C with phorbol 12-myristate 13-acetate (50 ng/ml; Sigma), ionomycin (750 ng/ml; Sigma) and brefeldin A (1.0 μg/ml; Sigma).

    Techniques: Isolation, Flow Cytometry, Cytometry, Staining

    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

    HRS is required for Vpu-induced BST-2 degradation and cell surface down-regulation. ( A–B ) HRS depletion prevents Vpu from targeting BST-2 for degradation. ( A ) HeLa cells transfected with either control siRNA (CT) or siRNA targeting HRS were infected with either VSV-G pseudotyped wt NL4-3 HIV-1 (NL4-3 WT) or VSV-G pseudotyped Vpu-defective NL4-3 (NL4-3 Udel) viruses at a MOI of 1, or left non-infected (NI). Forty-eight hours after infection, some of the cells were incubated with cycloheximide for 4 h and lysed. Cell lysates were analysed by western-blot. Tubulin is the loading control. These data are representative of 2 independent experiments. ( B ) HeLa cells transfected with either control siRNA (CT) or siRNA targeting HRS were infected with VSV-G pseudotyped NL4-3 HIV-1 (NL4-3 WT) and processed for immunolabelling with mouse polyclonal BST-2 and human HIV-1 Env (2G12) antibodies. Cells were imaged by confocal microscopy. Env staining discriminates between infected cells (arrows) and non-infected cells (arrowheads). Bar: 10 µm. ( C–D ) HRS depletion impairs Vpu-induced cell surface down regulation of BST-2. HeLa cells transfected with either control siRNA (CT) or siRNA targeting HRS were infected with VSV-G pseudotyped NL4-3 HIV-1 at a MOI of 0.5. Forty-eight hours later the cells were stained with rabbit polyclonal antibody against BST-2 (BST-2; upper panels) or rabbit polyclonal IgGs as an isotype control (CT IgG; lower panels), followed by a staining with a Cy5-conjugated donkey anti-rabbit antibody. The cells were then fixed, permeabilized and stained for Gag using a FITC-conjugated mouse monoclonal anti-CAp24. The cells were then processed for flow cytometry analysis. ( C ) Dot plot. Vertical lines indicate the gates set using non-infected cells stained as indicated. Gate B1 represents non infected cells and gate B2: infected-cells. ( D ) Bar graph representation of cell surface level of BST-2 in CAp24 negative cells (black bars) and CAp24 positive cells (grey bars) for each siRNA condition. Values are expressed as the Mean Fluorescence Intensity (MFI) on gate B1 (black bars) and B2 (grey bars) respectively. Error bars represent the –/+ SD from 3 independent experiments.

    Journal: PLoS Pathogens

    Article Title: The ESCRT-0 Component HRS is Required for HIV-1 Vpu-Mediated BST-2/Tetherin Down-Regulation

    doi: 10.1371/journal.ppat.1001265

    Figure Lengend Snippet: HRS is required for Vpu-induced BST-2 degradation and cell surface down-regulation. ( A–B ) HRS depletion prevents Vpu from targeting BST-2 for degradation. ( A ) HeLa cells transfected with either control siRNA (CT) or siRNA targeting HRS were infected with either VSV-G pseudotyped wt NL4-3 HIV-1 (NL4-3 WT) or VSV-G pseudotyped Vpu-defective NL4-3 (NL4-3 Udel) viruses at a MOI of 1, or left non-infected (NI). Forty-eight hours after infection, some of the cells were incubated with cycloheximide for 4 h and lysed. Cell lysates were analysed by western-blot. Tubulin is the loading control. These data are representative of 2 independent experiments. ( B ) HeLa cells transfected with either control siRNA (CT) or siRNA targeting HRS were infected with VSV-G pseudotyped NL4-3 HIV-1 (NL4-3 WT) and processed for immunolabelling with mouse polyclonal BST-2 and human HIV-1 Env (2G12) antibodies. Cells were imaged by confocal microscopy. Env staining discriminates between infected cells (arrows) and non-infected cells (arrowheads). Bar: 10 µm. ( C–D ) HRS depletion impairs Vpu-induced cell surface down regulation of BST-2. HeLa cells transfected with either control siRNA (CT) or siRNA targeting HRS were infected with VSV-G pseudotyped NL4-3 HIV-1 at a MOI of 0.5. Forty-eight hours later the cells were stained with rabbit polyclonal antibody against BST-2 (BST-2; upper panels) or rabbit polyclonal IgGs as an isotype control (CT IgG; lower panels), followed by a staining with a Cy5-conjugated donkey anti-rabbit antibody. The cells were then fixed, permeabilized and stained for Gag using a FITC-conjugated mouse monoclonal anti-CAp24. The cells were then processed for flow cytometry analysis. ( C ) Dot plot. Vertical lines indicate the gates set using non-infected cells stained as indicated. Gate B1 represents non infected cells and gate B2: infected-cells. ( D ) Bar graph representation of cell surface level of BST-2 in CAp24 negative cells (black bars) and CAp24 positive cells (grey bars) for each siRNA condition. Values are expressed as the Mean Fluorescence Intensity (MFI) on gate B1 (black bars) and B2 (grey bars) respectively. Error bars represent the –/+ SD from 3 independent experiments.

    Article Snippet: Flow cytometry Forty eight hours post-infection, siRNA transfected HeLa cells were harvested by scrapping, washed twice in cold PBS/1% (w/v) BSA and stained for 1 hour at 4°C with rabbit anti-BST-2 antibody (NIH) or control rabbit IgGs (rabbit serum; Sigma, France) in PBS/1%BSA.

    Techniques: Transfection, Infection, Incubation, Western Blot, Confocal Microscopy, Staining, Flow Cytometry, Cytometry, Fluorescence

    Overexpression of a dominant negative VPS4 mutant impairs Vpu-induced BST-2 cell surface down-regulation. ( A ) HeLa cells were transfected with plasmids encoding GFP-VPS4 or GFP-VPS4-E 223 /Q along with plasmids encoding the provirus NL4-3 wt (red and purple histograms) or Vpu-defective NL4-3 (NL4-3 Udel; blue and black histograms). Forty-eight hours later, the cells were co-stained with a mouse monoclonal antibody against BST-2 (BST-2; upper panels) or isotype control mouse IgG1 (CT Isotype; lower panels) and a human monoclonal antibody against Env. Cells were then co-stained with Alexa 647-conjugated donkey anti-mouse and PE-conjugated donkey anti-human antibodies, fixed, and processed for flow cytometry analysis. Histograms represent the relative cell number vs. BST-2 or Isotype IgG fluorescence intensity for the GFP HIV-1 positive cells (GFP + PE positive cells). Dotted histograms are the isotype controls. This experiment is representative of 3 independent experiments. ( B ) Bar graph representation of cell surface BST-2 in cell expressing (i) GFP-VPS4 wt along with HIV-1 NL4-3 WT (red bar) or HIV-1 NL4-3 Udel (blue bar) or (ii) GFP-VPS4-E 223 /Q CAp24 along with HIV-1 NL4-3 WT (purple bar) or HIV-1 NL4-3 Udel (black bar). Values are expressed as the Mean Fluorescence Intensity (MFI) obtained for BST-2 staining minus MFI values obtained for the corresponding isotype control. Error bars represent the –/+ SD from 3 independent experiments.

    Journal: PLoS Pathogens

    Article Title: The ESCRT-0 Component HRS is Required for HIV-1 Vpu-Mediated BST-2/Tetherin Down-Regulation

    doi: 10.1371/journal.ppat.1001265

    Figure Lengend Snippet: Overexpression of a dominant negative VPS4 mutant impairs Vpu-induced BST-2 cell surface down-regulation. ( A ) HeLa cells were transfected with plasmids encoding GFP-VPS4 or GFP-VPS4-E 223 /Q along with plasmids encoding the provirus NL4-3 wt (red and purple histograms) or Vpu-defective NL4-3 (NL4-3 Udel; blue and black histograms). Forty-eight hours later, the cells were co-stained with a mouse monoclonal antibody against BST-2 (BST-2; upper panels) or isotype control mouse IgG1 (CT Isotype; lower panels) and a human monoclonal antibody against Env. Cells were then co-stained with Alexa 647-conjugated donkey anti-mouse and PE-conjugated donkey anti-human antibodies, fixed, and processed for flow cytometry analysis. Histograms represent the relative cell number vs. BST-2 or Isotype IgG fluorescence intensity for the GFP HIV-1 positive cells (GFP + PE positive cells). Dotted histograms are the isotype controls. This experiment is representative of 3 independent experiments. ( B ) Bar graph representation of cell surface BST-2 in cell expressing (i) GFP-VPS4 wt along with HIV-1 NL4-3 WT (red bar) or HIV-1 NL4-3 Udel (blue bar) or (ii) GFP-VPS4-E 223 /Q CAp24 along with HIV-1 NL4-3 WT (purple bar) or HIV-1 NL4-3 Udel (black bar). Values are expressed as the Mean Fluorescence Intensity (MFI) obtained for BST-2 staining minus MFI values obtained for the corresponding isotype control. Error bars represent the –/+ SD from 3 independent experiments.

    Article Snippet: Flow cytometry Forty eight hours post-infection, siRNA transfected HeLa cells were harvested by scrapping, washed twice in cold PBS/1% (w/v) BSA and stained for 1 hour at 4°C with rabbit anti-BST-2 antibody (NIH) or control rabbit IgGs (rabbit serum; Sigma, France) in PBS/1%BSA.

    Techniques: Over Expression, Dominant Negative Mutation, Mutagenesis, Transfection, Staining, Flow Cytometry, Cytometry, Fluorescence, Expressing

    Design and analysis of a human 3D in vitro model of the breast tumor microenvironment A ) Schematic of a physiologically relevant human 3D in vitro model of the breast tumor microenvironment. Fluorescently-labeled human breast tumor cells and human mammary fibroblasts are incorporated in a Collagen I matrix seeded atop the porous membrane (8 μm pore size) of a tissue culture insert (inset-top: fibroblasts (red), tumor cells (green), nuclei (DAPI-blue). A confluent monolayer of human lymphatic endothelial cells is seeded on the alternate side of the membrane (inset bottom: CD31 (pink), nuclei (DAPI-blue)). B) Flow cytometry is used to identify ( B ) Cell Tracker-labeled breast tumor cells ( C ) uptake of live/dead indicator to assess cell death ( D ) Caspase 3/7 positivity to indicate apoptosis and (E) Track total cell death via apoptosis and necrosis . Negative controls are shown in gray on histogram plots.

    Journal: Methods (San Diego, Calif.)

    Article Title: Assessing multiparametric drug response in tissue engineered tumor microenvironment models

    doi: 10.1016/j.ymeth.2017.12.010

    Figure Lengend Snippet: Design and analysis of a human 3D in vitro model of the breast tumor microenvironment A ) Schematic of a physiologically relevant human 3D in vitro model of the breast tumor microenvironment. Fluorescently-labeled human breast tumor cells and human mammary fibroblasts are incorporated in a Collagen I matrix seeded atop the porous membrane (8 μm pore size) of a tissue culture insert (inset-top: fibroblasts (red), tumor cells (green), nuclei (DAPI-blue). A confluent monolayer of human lymphatic endothelial cells is seeded on the alternate side of the membrane (inset bottom: CD31 (pink), nuclei (DAPI-blue)). B) Flow cytometry is used to identify ( B ) Cell Tracker-labeled breast tumor cells ( C ) uptake of live/dead indicator to assess cell death ( D ) Caspase 3/7 positivity to indicate apoptosis and (E) Track total cell death via apoptosis and necrosis . Negative controls are shown in gray on histogram plots.

    Article Snippet: Flow cytometry plots were generated using Millipore Easycyte software.

    Techniques: In Vitro, Labeling, Flow Cytometry, Cytometry

    Implementation of tumor microenvironment models to examine response to chemotherapy at the tumor-stroma interface The tumor microenvironment is composed of not only tumor cells, but also stromal, immune, and endothelial cells, as well as extracellular matrix and fluid flow. We use tissue-engineering to mimic specific components of this region. A) We label our cell populations with cell tracker dyes, incorporate them in a relevant extracellular matrix, and B) seed into tissue culture inserts. After gels are set, C) chemotherapeutics can be dosed across the gels. After 48 hours of dosing and flushing of system, D) gels can be removed, the matrix degraded, and the remaining cells can be analyzed via E) flow cytometry and fluorescence microscopy.

    Journal: Methods (San Diego, Calif.)

    Article Title: Assessing multiparametric drug response in tissue engineered tumor microenvironment models

    doi: 10.1016/j.ymeth.2017.12.010

    Figure Lengend Snippet: Implementation of tumor microenvironment models to examine response to chemotherapy at the tumor-stroma interface The tumor microenvironment is composed of not only tumor cells, but also stromal, immune, and endothelial cells, as well as extracellular matrix and fluid flow. We use tissue-engineering to mimic specific components of this region. A) We label our cell populations with cell tracker dyes, incorporate them in a relevant extracellular matrix, and B) seed into tissue culture inserts. After gels are set, C) chemotherapeutics can be dosed across the gels. After 48 hours of dosing and flushing of system, D) gels can be removed, the matrix degraded, and the remaining cells can be analyzed via E) flow cytometry and fluorescence microscopy.

    Article Snippet: Flow cytometry plots were generated using Millipore Easycyte software.

    Techniques: Flow Cytometry, Cytometry, Fluorescence, Microscopy

    Design and analysis of a human 3D in vitro model of the brain tumor microenvironment A) Glioma microenvironment model comprising human glioma cells (red), human astrocytes (green), and human microglia (purple) in a hyaluronan matrix (top inset). Invasion of Cell Tracker labeled U251 glioma cells through the porous (8 μm pore size) membrane (DAPI-blue) is analyzed using fluorescence microscopy (bottom inset). B) Flow cytometry is used to identify ( B ) Cell Tracker- labeled U251 glioma cells, ( C ) uptake of live/dead indicator to assess cell death, ( D ) Ki67 expression to assess proliferation, and ( E ) CD71 expression to assess stem-like cells. Negative controls are shown in gray on histogram plots.

    Journal: Methods (San Diego, Calif.)

    Article Title: Assessing multiparametric drug response in tissue engineered tumor microenvironment models

    doi: 10.1016/j.ymeth.2017.12.010

    Figure Lengend Snippet: Design and analysis of a human 3D in vitro model of the brain tumor microenvironment A) Glioma microenvironment model comprising human glioma cells (red), human astrocytes (green), and human microglia (purple) in a hyaluronan matrix (top inset). Invasion of Cell Tracker labeled U251 glioma cells through the porous (8 μm pore size) membrane (DAPI-blue) is analyzed using fluorescence microscopy (bottom inset). B) Flow cytometry is used to identify ( B ) Cell Tracker- labeled U251 glioma cells, ( C ) uptake of live/dead indicator to assess cell death, ( D ) Ki67 expression to assess proliferation, and ( E ) CD71 expression to assess stem-like cells. Negative controls are shown in gray on histogram plots.

    Article Snippet: Flow cytometry plots were generated using Millipore Easycyte software.

    Techniques: In Vitro, Labeling, Fluorescence, Microscopy, Flow Cytometry, Cytometry, Expressing

    Detection of chemotherapeutic efficacy and cellular uptake of doxorubicin in a breast-mimetic human 3D in vitro system A ) Viability of of human breast tumor cell line HCC38 in our 3D in vitro breast tumor microenvironment model in response to increasing concentrations of doxorubicin (0.01–10 μM) as assessed by flow cytometry (% live/dead+ cell tracker+). B ) Viability response of breast tumor cell line HCC1806. C) Invasion of human breast tumor cell line HCC 38 after treatment with increasing concentrations of doxorubicin (0.01–10 μM) as assessed by fluorescent imaging of tissue culture insert membranes. D ) Invasion response of breast tumor cell line HCC1806. E ) Cellular uptake of increasing concentrations of doxorubicin by HCC38 tumor cells indicating shifting Mean Fluorescence Intensity (x-axis). Cellular uptake of increasing concentrations of doxorubicin by human mammary fibroblasts indicating shifting Mean Fluorescence Intensity (x-axis). * p

    Journal: Methods (San Diego, Calif.)

    Article Title: Assessing multiparametric drug response in tissue engineered tumor microenvironment models

    doi: 10.1016/j.ymeth.2017.12.010

    Figure Lengend Snippet: Detection of chemotherapeutic efficacy and cellular uptake of doxorubicin in a breast-mimetic human 3D in vitro system A ) Viability of of human breast tumor cell line HCC38 in our 3D in vitro breast tumor microenvironment model in response to increasing concentrations of doxorubicin (0.01–10 μM) as assessed by flow cytometry (% live/dead+ cell tracker+). B ) Viability response of breast tumor cell line HCC1806. C) Invasion of human breast tumor cell line HCC 38 after treatment with increasing concentrations of doxorubicin (0.01–10 μM) as assessed by fluorescent imaging of tissue culture insert membranes. D ) Invasion response of breast tumor cell line HCC1806. E ) Cellular uptake of increasing concentrations of doxorubicin by HCC38 tumor cells indicating shifting Mean Fluorescence Intensity (x-axis). Cellular uptake of increasing concentrations of doxorubicin by human mammary fibroblasts indicating shifting Mean Fluorescence Intensity (x-axis). * p

    Article Snippet: Flow cytometry plots were generated using Millipore Easycyte software.

    Techniques: In Vitro, Flow Cytometry, Cytometry, Imaging, Fluorescence

    A genetic screen for regulators of TORC1. (A) A schematic of the TORC1 pathway. In nutrient rich environment, TORC1 is active and Gln3/Gat1 are in the cytoplasm. Upon TORC1 inactivation by rapamycin or amino acid starvation, Gln3 and Gat1 translocate into the nucleus, leading to the transcriptional activation of Dal80. (B) Dal80 gene expression reporter contains 600 basepairs of Dal80 promoter driving the expression of GFP. HygB, hygromycin B resistance gene, is used for selection of transformants. (C) Accumulation of GFP in cells is linear. Wildtype cells transformed with Dal80pr-GFP plasmid were treated either with rapamycin (20 ng/mL) or inoculated into nitrogen free medium. At indicated timepoints the average GFP content of the cells was determined by flow cytometry. (D) Fifty-five 96-well plates, containing all non-essential yeast deletion strains, were transformed with the Dal80pr-GFP reporter plasmid. The cells in each plate were grown under 3 conditions: YPD for 6 hours, YPD+rapamycin for 15 hours or N-free media for 4 hours. GFP content was determined by flow cytometry.

    Journal: PLoS Genetics

    Article Title: A Genome-Wide Screen for Regulators of TORC1 in Response to Amino Acid Starvation Reveals a Conserved Npr2/3 Complex

    doi: 10.1371/journal.pgen.1000515

    Figure Lengend Snippet: A genetic screen for regulators of TORC1. (A) A schematic of the TORC1 pathway. In nutrient rich environment, TORC1 is active and Gln3/Gat1 are in the cytoplasm. Upon TORC1 inactivation by rapamycin or amino acid starvation, Gln3 and Gat1 translocate into the nucleus, leading to the transcriptional activation of Dal80. (B) Dal80 gene expression reporter contains 600 basepairs of Dal80 promoter driving the expression of GFP. HygB, hygromycin B resistance gene, is used for selection of transformants. (C) Accumulation of GFP in cells is linear. Wildtype cells transformed with Dal80pr-GFP plasmid were treated either with rapamycin (20 ng/mL) or inoculated into nitrogen free medium. At indicated timepoints the average GFP content of the cells was determined by flow cytometry. (D) Fifty-five 96-well plates, containing all non-essential yeast deletion strains, were transformed with the Dal80pr-GFP reporter plasmid. The cells in each plate were grown under 3 conditions: YPD for 6 hours, YPD+rapamycin for 15 hours or N-free media for 4 hours. GFP content was determined by flow cytometry.

    Article Snippet: All flow cytometry readings were conducted on the Guava PCA-96 AFP system (Guava Technologies, Hayward, CA).

    Techniques: Activation Assay, Expressing, Selection, Transformation Assay, Plasmid Preparation, Flow Cytometry, Cytometry

    Exposure of cell surface components is altered in pfa4 Δ cells. (A) Example of flow cytometry profiles used to assess the exposure/accessibility of cell wall components. Fluorescence intensity profiles of H99 and chs3 Δ cells, either unstained (gray) or stained with calcofluor white (CFW; light blue) are overlaid to illustrate the difference in mean fluorescence intensity (ΔMFI). (B) ΔMFI for staining with CFW (binds chitin), Eosin Y (binds chitosan), Concanavalin A (binds mannoproteins), LY, and Pont (bind unspecified cell wall components); mean ± SEM of three independent experiments, with values normalized to the highest bar for each strain.

    Journal: PLoS Pathogens

    Article Title: A Single Protein S-acyl Transferase Acts through Diverse Substrates to Determine Cryptococcal Morphology, Stress Tolerance, and Pathogenic Outcome

    doi: 10.1371/journal.ppat.1004908

    Figure Lengend Snippet: Exposure of cell surface components is altered in pfa4 Δ cells. (A) Example of flow cytometry profiles used to assess the exposure/accessibility of cell wall components. Fluorescence intensity profiles of H99 and chs3 Δ cells, either unstained (gray) or stained with calcofluor white (CFW; light blue) are overlaid to illustrate the difference in mean fluorescence intensity (ΔMFI). (B) ΔMFI for staining with CFW (binds chitin), Eosin Y (binds chitosan), Concanavalin A (binds mannoproteins), LY, and Pont (bind unspecified cell wall components); mean ± SEM of three independent experiments, with values normalized to the highest bar for each strain.

    Article Snippet: Fluorescence microscopy and flow cytometry Cells were grown overnight at 30°C in YPD (with appropriate antibiotics if needed to maintain plasmids), diluted as for phenotyping, washed in PBS, and resuspended at 107 /mL for staining as follows (all manipulations at RT): For LY and EoY (Sigma), cells were washed once in McIlvaines buffer, pH 6.0; resuspended in the same; and incubated for ~15 min with 250 μg/mL of the dye.

    Techniques: Flow Cytometry, Cytometry, Fluorescence, Staining

    HAdV-D37 binding, entry, early gene expression, and DNA replication in the MyD88 −/− corneas and human corneal fibroblasts treated with a MyD88 inhibitor peptide. Corneas of WT and MyD88 −/− mice injected with Cy3-labeled HAdV-D37 (red) and visualized by confocal microscopy at 1 hpi ( A ) appear to show binding to corneal stromal cells of both mouse genotypes. Slides were co-stained with Topro-3 and phalloidin to visualize nuclei (blue) and cytosolic actin filaments (green). Flow cytometry performed at 1 hpi ( B ) shows no difference in viral binding to cultured HCF pretreated with the MyD88 inhibitory peptide or the control peptide (Pepinh-MyD, Pepinh-Ctl, respectively) prior to infection with Cy3 labeled virus. Real-time qPCR ( C ) performed at 1 dpi on mouse corneas shows no significant difference in the quantity of viral DNA in MyD88 −/− corneas as compared to those of WT mice (p=.21). However, real-time qRT-PCR ( D ) for early viral gene expression shows a significant reduction in E1A and E1B expression in MyD88 −/− as compared to WT corneas (*p=.00167 and *p

    Journal: Immunology and cell biology

    Article Title: Role of MyD88 in Adenovirus Keratitis

    doi: 10.1038/icb.2016.73

    Figure Lengend Snippet: HAdV-D37 binding, entry, early gene expression, and DNA replication in the MyD88 −/− corneas and human corneal fibroblasts treated with a MyD88 inhibitor peptide. Corneas of WT and MyD88 −/− mice injected with Cy3-labeled HAdV-D37 (red) and visualized by confocal microscopy at 1 hpi ( A ) appear to show binding to corneal stromal cells of both mouse genotypes. Slides were co-stained with Topro-3 and phalloidin to visualize nuclei (blue) and cytosolic actin filaments (green). Flow cytometry performed at 1 hpi ( B ) shows no difference in viral binding to cultured HCF pretreated with the MyD88 inhibitory peptide or the control peptide (Pepinh-MyD, Pepinh-Ctl, respectively) prior to infection with Cy3 labeled virus. Real-time qPCR ( C ) performed at 1 dpi on mouse corneas shows no significant difference in the quantity of viral DNA in MyD88 −/− corneas as compared to those of WT mice (p=.21). However, real-time qRT-PCR ( D ) for early viral gene expression shows a significant reduction in E1A and E1B expression in MyD88 −/− as compared to WT corneas (*p=.00167 and *p

    Article Snippet: Flow Cytometry Corneas were dissected from mouse eyes at 4 days post infection (dpi) and cut into small (1–2 mm diameter) fragments for subsequent digestion with 1 mg/ml collagenase type I and 0.5 mg/ml DNase (Sigma Chemical, St. Louis, MO) .

    Techniques: Binding Assay, Expressing, Mouse Assay, Injection, Labeling, Confocal Microscopy, Staining, Flow Cytometry, Cytometry, Cell Culture, CTL Assay, Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    MyD88 −/− keratitis after HAdV-D37 infection. The corneas of WT C57BL/6j and MyD88 −/− mice on a C57BL/6j background were injected intrastromally with 1 µl of 10 5 TCID 50 HAdV-D37 or virus-free dialysis buffer control. Representative clinical photographs ( A ) and histology with H E staining ( B ), both at 4 dpi, show central corneal opacity and stromal infiltration, respectively, in infected WT corneas as compared to MyD88 −/− corneas. Flow cytometry ( C ) performed 4 dpi for CD45 + cell infiltration shows significantly more CD45 + events in infected WT than in similarly infected MyD88 −/− corneas (*p

    Journal: Immunology and cell biology

    Article Title: Role of MyD88 in Adenovirus Keratitis

    doi: 10.1038/icb.2016.73

    Figure Lengend Snippet: MyD88 −/− keratitis after HAdV-D37 infection. The corneas of WT C57BL/6j and MyD88 −/− mice on a C57BL/6j background were injected intrastromally with 1 µl of 10 5 TCID 50 HAdV-D37 or virus-free dialysis buffer control. Representative clinical photographs ( A ) and histology with H E staining ( B ), both at 4 dpi, show central corneal opacity and stromal infiltration, respectively, in infected WT corneas as compared to MyD88 −/− corneas. Flow cytometry ( C ) performed 4 dpi for CD45 + cell infiltration shows significantly more CD45 + events in infected WT than in similarly infected MyD88 −/− corneas (*p

    Article Snippet: Flow Cytometry Corneas were dissected from mouse eyes at 4 days post infection (dpi) and cut into small (1–2 mm diameter) fragments for subsequent digestion with 1 mg/ml collagenase type I and 0.5 mg/ml DNase (Sigma Chemical, St. Louis, MO) .

    Techniques: Infection, Mouse Assay, Injection, Staining, Flow Cytometry, Cytometry

    Cytokine responses of lung T cells to antigen 85A. Mice were immunized with Ad85A i.n. (Ad85A i.n.) or primed with BCG and boosted 8 weeks later with Ad85A intranasally (B-Ad85A i.n.). Lung cells were isolated at the indicated times after the last immunization and stimulated with pooled 85A peptides for 6 hours. The frequencies of IFNγ, IL-2 and TNFα producing cells were determined by flow cytometry on CD8 (A) and CD4 (B) gated cells. Pie charts indicate the proportions of single (light grey), dual (dark grey) and triple (black) CD8 producers of IFNγ, TNFα and IL-2 in the lungs of mice 4 (C) and 23 (D) weeks after immunization. Results are expressed as the mean +/− SEM of three/four mice per group, representative of two independent experiments.

    Journal: PLoS ONE

    Article Title: Immunization of Mice with a Recombinant Adenovirus Vaccine Inhibits the Early Growth of Mycobacterium tuberculosis After Infection

    doi: 10.1371/journal.pone.0008235

    Figure Lengend Snippet: Cytokine responses of lung T cells to antigen 85A. Mice were immunized with Ad85A i.n. (Ad85A i.n.) or primed with BCG and boosted 8 weeks later with Ad85A intranasally (B-Ad85A i.n.). Lung cells were isolated at the indicated times after the last immunization and stimulated with pooled 85A peptides for 6 hours. The frequencies of IFNγ, IL-2 and TNFα producing cells were determined by flow cytometry on CD8 (A) and CD4 (B) gated cells. Pie charts indicate the proportions of single (light grey), dual (dark grey) and triple (black) CD8 producers of IFNγ, TNFα and IL-2 in the lungs of mice 4 (C) and 23 (D) weeks after immunization. Results are expressed as the mean +/− SEM of three/four mice per group, representative of two independent experiments.

    Article Snippet: Cell Isolation and Flow Cytometry Lungs were perfused with PBS, minced and digested with 0.7 mg/ml collagenase type 1 (Sigma) and 30 µg/ml DNase 1 (Sigma) for 45 mins at 37°C, then passed through a cell strainer, washed and monuclear cells selected by density centrifugation on Lympholyte (Cederlane).

    Techniques: Mouse Assay, Isolation, Flow Cytometry, Cytometry

    Combination RT and anti-CD40 leads to increased intratumoral CD8 T cells. (A) Tumors from mice treated with 5Gy SBRT and/or a single dose of anti-CD40 (20 μg) were harvested at 2 weeks post-treatment and analyzed by immunohistochemistry for CD8. (B) Quantification of a. n = 5 mice/group. (C) Tumors from mice treated as in Figure 2 were harvested at 2 weeks post-treatment, digested and analyzed by flow cytometry. GrMDSCs: CD11b + Gr1 high ; MoMDSCs: CD11b + Ly6C + ; macrophages: CD11b + ,Gr1 − . (D) Ratio of CD8 T cells to total CD11b + myeloid cells. n = 5 mice/group. Error bars are SD. * p

    Journal: Frontiers in Immunology

    Article Title: Radiation and Local Anti-CD40 Generate an Effective in situ Vaccine in Preclinical Models of Pancreatic Cancer

    doi: 10.3389/fimmu.2018.02030

    Figure Lengend Snippet: Combination RT and anti-CD40 leads to increased intratumoral CD8 T cells. (A) Tumors from mice treated with 5Gy SBRT and/or a single dose of anti-CD40 (20 μg) were harvested at 2 weeks post-treatment and analyzed by immunohistochemistry for CD8. (B) Quantification of a. n = 5 mice/group. (C) Tumors from mice treated as in Figure 2 were harvested at 2 weeks post-treatment, digested and analyzed by flow cytometry. GrMDSCs: CD11b + Gr1 high ; MoMDSCs: CD11b + Ly6C + ; macrophages: CD11b + ,Gr1 − . (D) Ratio of CD8 T cells to total CD11b + myeloid cells. n = 5 mice/group. Error bars are SD. * p

    Article Snippet: Flow cytometry Tumors were extracted from mice, digested in RPMI supplemented with type II collagenase (Sigma) and soybean trypsin inhibitor (Life Technologies), and dispersed into a single-cell suspension by filtering with a 40 micron cell strainer.

    Techniques: Mouse Assay, Immunohistochemistry, Flow Cytometry, Cytometry

    Inhibition of PI3K activity results in the apoptosis of CEF cells ( A ) CEF cells were preincubated with the PI3K inhibitor LY294002 (LY) (20 μM) or equal volumes of DMSO for 1 h. They were then infected with NDV-GM at an MOI of 1, and harvested and lysed at 0, 12, 18, and 24 hpi. Specific antibodies were used to assess the phosphorylation of Akt (Ser 473) and GSK-3β, as well as the cleavage of caspase-3 and PARP by western blot. ( B ) CEF cells were pretreated with the specific PI3K inhibitor LY294002 (20 μM) or equal volumes of DMSO for 1 h. After being infected with NDV-GM strain, they were harvested for annexin V apoptosis detection and PI double staining. Then they were analyzed by flow cytometry at 12, 24, and 48 h postinfection (hpi). ( C ) The number of apoptosis cells ( M ± SD of triplicates) are quantified in (B); * p

    Journal: Oncotarget

    Article Title: Transient activation of the PI3K/Akt pathway promotes Newcastle disease virus replication and enhances anti-apoptotic signaling responses

    doi: 10.18632/oncotarget.15796

    Figure Lengend Snippet: Inhibition of PI3K activity results in the apoptosis of CEF cells ( A ) CEF cells were preincubated with the PI3K inhibitor LY294002 (LY) (20 μM) or equal volumes of DMSO for 1 h. They were then infected with NDV-GM at an MOI of 1, and harvested and lysed at 0, 12, 18, and 24 hpi. Specific antibodies were used to assess the phosphorylation of Akt (Ser 473) and GSK-3β, as well as the cleavage of caspase-3 and PARP by western blot. ( B ) CEF cells were pretreated with the specific PI3K inhibitor LY294002 (20 μM) or equal volumes of DMSO for 1 h. After being infected with NDV-GM strain, they were harvested for annexin V apoptosis detection and PI double staining. Then they were analyzed by flow cytometry at 12, 24, and 48 h postinfection (hpi). ( C ) The number of apoptosis cells ( M ± SD of triplicates) are quantified in (B); * p

    Article Snippet: Flow cytometry Apoptosis was detected by flow cytometry using Annexin V-FITC/PI Apoptosis Detection Kit (Sigma–Aldrich), following the manufacturer's instructions.

    Techniques: Inhibition, Activity Assay, Infection, Western Blot, Double Staining, Flow Cytometry, Cytometry

    PI3K signaling pathway and apoptosis are simultaneously involved in NDV replication CEF cells were preincubated with the pan caspase inhibitor ZVAD-FMK (40 μM) for 1 h, with or without PI3K inhibitor LY294002 (LY) (20 μM) prior to infection with NDV-GM at an MOI of 1 for 48 h. ( A ) Cell death was analyzed under microscope and by MTT assay. ( B ) The number of apoptotic cells was calculated by flow cytometry using Annexin V-FITC/PI Apoptosis Detection Kit. * p

    Journal: Oncotarget

    Article Title: Transient activation of the PI3K/Akt pathway promotes Newcastle disease virus replication and enhances anti-apoptotic signaling responses

    doi: 10.18632/oncotarget.15796

    Figure Lengend Snippet: PI3K signaling pathway and apoptosis are simultaneously involved in NDV replication CEF cells were preincubated with the pan caspase inhibitor ZVAD-FMK (40 μM) for 1 h, with or without PI3K inhibitor LY294002 (LY) (20 μM) prior to infection with NDV-GM at an MOI of 1 for 48 h. ( A ) Cell death was analyzed under microscope and by MTT assay. ( B ) The number of apoptotic cells was calculated by flow cytometry using Annexin V-FITC/PI Apoptosis Detection Kit. * p

    Article Snippet: Flow cytometry Apoptosis was detected by flow cytometry using Annexin V-FITC/PI Apoptosis Detection Kit (Sigma–Aldrich), following the manufacturer's instructions.

    Techniques: Infection, Microscopy, MTT Assay, Flow Cytometry, Cytometry

    Cancer stem-like cells can arise as a result of EMT A. Cancer stem cell–related marker expression in mammosphere cultured cells. Cell lysates from adherent cells and mammosphere cultured cells were analyzed by Western blot with indicated antibodies. B. Mammosphere cultured cells show CD44 high/CD24 low expression at the cell surface. Cells were cytospun onto glass slides for CD44 and CD24 antibody staining. Representative images were taken by fluorescence microscopy. C. Analysis using flow cytometry further revealed that sphere cells exhibited an increase in CD44 high/CD24 low populations.

    Journal: Oncotarget

    Article Title: Epithelial-to-mesenchymal transition leads to loss of EpCAM and different physical properties in circulating tumor cells from metastatic breast cancer

    doi: 10.18632/oncotarget.8250

    Figure Lengend Snippet: Cancer stem-like cells can arise as a result of EMT A. Cancer stem cell–related marker expression in mammosphere cultured cells. Cell lysates from adherent cells and mammosphere cultured cells were analyzed by Western blot with indicated antibodies. B. Mammosphere cultured cells show CD44 high/CD24 low expression at the cell surface. Cells were cytospun onto glass slides for CD44 and CD24 antibody staining. Representative images were taken by fluorescence microscopy. C. Analysis using flow cytometry further revealed that sphere cells exhibited an increase in CD44 high/CD24 low populations.

    Article Snippet: Flow cytometry Adherent and sphere cells were detached with accutase (Sigma) and incubated in staining buffer with anti-EpCAM (PE-conjugated), anti-CD44 (APC-conjugated) and anti-CD24 (FITC-conjugated) and finally analyzed by flow cytometry.

    Techniques: Marker, Expressing, Cell Culture, Western Blot, Staining, Fluorescence, Microscopy, Flow Cytometry, Cytometry

    Staining of BxPC-3 cells with anti-CEA antibody and quantification of binding sites. BxPC-3 cells were treated with anti-CEA antibody and stained with a fluorochrome conjugated secondary antibody against anti-CEA (red, filled), or control-stained with secondary antibody only (black, open) and analyzed by flow cytometry. Gating was for the main population (A). The anti-CEA antibody binding capacity, determined with reference beads, was very high (2,227,000 binding sites per cell) (B).

    Journal: PLoS ONE

    Article Title: Near Infra-Red Photoimmunotherapy with Anti-CEA-IR700 Results in Extensive Tumor Lysis and a Significant Decrease in Tumor Burden in Orthotopic Mouse Models of Pancreatic Cancer

    doi: 10.1371/journal.pone.0121989

    Figure Lengend Snippet: Staining of BxPC-3 cells with anti-CEA antibody and quantification of binding sites. BxPC-3 cells were treated with anti-CEA antibody and stained with a fluorochrome conjugated secondary antibody against anti-CEA (red, filled), or control-stained with secondary antibody only (black, open) and analyzed by flow cytometry. Gating was for the main population (A). The anti-CEA antibody binding capacity, determined with reference beads, was very high (2,227,000 binding sites per cell) (B).

    Article Snippet: Flow cytometry profiles from the anti-CEA antibody-treated cells and the untreated cells were established on a Guava EasyCyte Plus flow cytometer (EMD Millipore, Billerica, MA).

    Techniques: Staining, Binding Assay, Flow Cytometry, Cytometry

    PTX therapy rescued TCR expression and influenced naïve/memory/activation phenotypes of CD8 + T-cells. (A) Flow cytometry analysis of CD8 + T-cells in the spleen. (B) Frequency of TCRαβ Low cells and mean fluorescence intensity (MFI) of TCR in CD8 + T-cells. (C) Frequencies of splenic TCR + CD8 + cells expressing CD45RA + CCR7 + (naïve), CD45RA - CCR7 + (central memory) and CD45RA - CCR7 - (effector memory). (D) Frequencies of CD44 - CD62L + (naïve), CD44 + CD62L + (central memory) and CD44 + CD62L - (activated) CD8 + T-cells. The results represent three to five mice per experimental group in three independent experiments. ** p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Pentoxifylline Reverses Chronic Experimental Chagasic Cardiomyopathy in Association with Repositioning of Abnormal CD8+ T-Cell Response

    doi: 10.1371/journal.pntd.0003659

    Figure Lengend Snippet: PTX therapy rescued TCR expression and influenced naïve/memory/activation phenotypes of CD8 + T-cells. (A) Flow cytometry analysis of CD8 + T-cells in the spleen. (B) Frequency of TCRαβ Low cells and mean fluorescence intensity (MFI) of TCR in CD8 + T-cells. (C) Frequencies of splenic TCR + CD8 + cells expressing CD45RA + CCR7 + (naïve), CD45RA - CCR7 + (central memory) and CD45RA - CCR7 - (effector memory). (D) Frequencies of CD44 - CD62L + (naïve), CD44 + CD62L + (central memory) and CD44 + CD62L - (activated) CD8 + T-cells. The results represent three to five mice per experimental group in three independent experiments. ** p

    Article Snippet: Flow cytometry analysis Spleens were minced and the red blood cells were removed using lysis buffer (Sigma-Aldrich, USA).

    Techniques: Expressing, Activation Assay, Flow Cytometry, Cytometry, Fluorescence, Mouse Assay

    Selected bivariate scatter plots of lung cell suspension in experimental groups by flow cytometry. Notes: Anti-CD31 and anti-CD45 antibodies were labeled with fluorescent APC and anti-Sca-1 antibody with fluorescent FITC. The percentage of CD31 − CD45 − Sca-1 + population in single-cell suspension from the whole lung is indicated by the polygon drawn by the black line. ( A ) Blank control without fluorescent antibody; ( B ) PBS group; ( C ) CSE group; ( D ) CSE + adenovirus group. Autofluorescence of lung tissues was also confirmed. Abbreviations: FITC, fluorescein isothiocyanate; FITC-A, antibody with fluorescein isothiocyanate; PBS, phosphate-buffered saline; CSE, cigarette smoke extract.

    Journal: International Journal of Chronic Obstructive Pulmonary Disease

    Article Title: Changes in the number of CD31−CD45−Sca-1+ cells and Shh signaling pathway involvement in the lungs of mice with emphysema and relevant effects of acute adenovirus infection

    doi: 10.2147/COPD.S129871

    Figure Lengend Snippet: Selected bivariate scatter plots of lung cell suspension in experimental groups by flow cytometry. Notes: Anti-CD31 and anti-CD45 antibodies were labeled with fluorescent APC and anti-Sca-1 antibody with fluorescent FITC. The percentage of CD31 − CD45 − Sca-1 + population in single-cell suspension from the whole lung is indicated by the polygon drawn by the black line. ( A ) Blank control without fluorescent antibody; ( B ) PBS group; ( C ) CSE group; ( D ) CSE + adenovirus group. Autofluorescence of lung tissues was also confirmed. Abbreviations: FITC, fluorescein isothiocyanate; FITC-A, antibody with fluorescein isothiocyanate; PBS, phosphate-buffered saline; CSE, cigarette smoke extract.

    Article Snippet: Flow cytometry quantification of lung CD31− CD45− Sca-1+ cells At day 28, the remaining mice (n=8 per group) were anesthetized with intraperitoneal injection of 10% chlorine hydrate (3 mL/kg), followed by right atrium perfusion with 10 mL PBS and endotracheal instillation of 3 mL collagenase IV (150 U/mL; Sigma-Aldrich, St Louis, MO, USA) and 0.3 mL 1% low melting point agarose.

    Techniques: Flow Cytometry, Cytometry, Labeling

    The role of ERK1/2 in LPS-mediated platelet potentiation. Washed human platelets stimulated or not with U46619 (0.25μM) in the presence or absence of LPS from E . coli O111:B4 (1μg/mL) were analysed by immunoblotting using antiphospho- ERK1/2 antibody. Total levels of 14-3-3 ζ were measured on each sample as a loading control (A). Human-washed platelet aggregation was performed by optical aggregometry following stimulation with U46619 (0.25μM) in the presence or absence of LPS from E . coli O111:B4 (1μg/mL) after 3 min of incubation with Cobimetinib (100μM) before activation with U46619 (0.25μM) (B). The effect of U46619 (0.25μM) and LPS- induced fibrinogen binding and P-selectin exposure after incubation with Cobimetinib (100μM) were measured in PRP by flow cytometry (C and D). Washed platelets (4 x 10 8 /mL) were pre-incubated with 10μM DCFH-DA in the presence or absence of Cobimetinib (100μM) before being activated with U46619 (0.25μM) in the presence or absence of LPS from E . coli O111:B4 (1μg/mL) and ROS levels were analysed by flow cytometry (E). Cumulative data represent mean values ± SEM (n = 4). (Anova-Bonferroni test, * P≤ 0.05; ** P≤ 0.01; *** P≤ 0.001; Test t student # P≤ 0.05; # # P≤ 0.01).

    Journal: PLoS ONE

    Article Title: Lipopolysaccharide potentiates platelet responses via toll-like receptor 4-stimulated Akt-Erk-PLA2 signalling

    doi: 10.1371/journal.pone.0186981

    Figure Lengend Snippet: The role of ERK1/2 in LPS-mediated platelet potentiation. Washed human platelets stimulated or not with U46619 (0.25μM) in the presence or absence of LPS from E . coli O111:B4 (1μg/mL) were analysed by immunoblotting using antiphospho- ERK1/2 antibody. Total levels of 14-3-3 ζ were measured on each sample as a loading control (A). Human-washed platelet aggregation was performed by optical aggregometry following stimulation with U46619 (0.25μM) in the presence or absence of LPS from E . coli O111:B4 (1μg/mL) after 3 min of incubation with Cobimetinib (100μM) before activation with U46619 (0.25μM) (B). The effect of U46619 (0.25μM) and LPS- induced fibrinogen binding and P-selectin exposure after incubation with Cobimetinib (100μM) were measured in PRP by flow cytometry (C and D). Washed platelets (4 x 10 8 /mL) were pre-incubated with 10μM DCFH-DA in the presence or absence of Cobimetinib (100μM) before being activated with U46619 (0.25μM) in the presence or absence of LPS from E . coli O111:B4 (1μg/mL) and ROS levels were analysed by flow cytometry (E). Cumulative data represent mean values ± SEM (n = 4). (Anova-Bonferroni test, * P≤ 0.05; ** P≤ 0.01; *** P≤ 0.001; Test t student # P≤ 0.05; # # P≤ 0.01).

    Article Snippet: Platelet ROS-production measured by flow cytometry Production of ROS by platelets was measured by flow cytometry following the loading of platelets with the fluorescent dye, 2’ ,7’ -dichlorofluorescin diacetate (DCFH-DA) (Sigma Aldrich, UK).

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

    ROS release induced by LPS promotes potentiation of aggregation. Washed platelets (4 x 10 8 /ml) were pre-incubated with 10μM DCFH-DA in the presence or absence of vehicle, LPS from E . coli O111:B4 (0.5 or 1μg/mL) or LPS RS (1μg/mL), superoxide dismutase- SOD (30U/mL) or catalase (300U/mL) before being activated with U46619 (0.25μM). Samples were analysed by flow cytometry and the levels of ROS released were detected and expressed as % increase above levels detected in unstimulated platelets (A and B). Human-washed platelet aggregation was performed by optical aggregometry following stimulation with U46619 (0.25μM) in the presence or absence of LPS from E . coli (1μg/mL) after 3 min of incubation with superoxide dismutase- SOD (30U/mL) or catalase (300U/mL) (C). Cumulative data represent mean values ± SEM (n = 4). (Anova-Bonferroni test, * P≤ 0.05; Test t student # P≤ 0.01; # # P≤ 0.01).

    Journal: PLoS ONE

    Article Title: Lipopolysaccharide potentiates platelet responses via toll-like receptor 4-stimulated Akt-Erk-PLA2 signalling

    doi: 10.1371/journal.pone.0186981

    Figure Lengend Snippet: ROS release induced by LPS promotes potentiation of aggregation. Washed platelets (4 x 10 8 /ml) were pre-incubated with 10μM DCFH-DA in the presence or absence of vehicle, LPS from E . coli O111:B4 (0.5 or 1μg/mL) or LPS RS (1μg/mL), superoxide dismutase- SOD (30U/mL) or catalase (300U/mL) before being activated with U46619 (0.25μM). Samples were analysed by flow cytometry and the levels of ROS released were detected and expressed as % increase above levels detected in unstimulated platelets (A and B). Human-washed platelet aggregation was performed by optical aggregometry following stimulation with U46619 (0.25μM) in the presence or absence of LPS from E . coli (1μg/mL) after 3 min of incubation with superoxide dismutase- SOD (30U/mL) or catalase (300U/mL) (C). Cumulative data represent mean values ± SEM (n = 4). (Anova-Bonferroni test, * P≤ 0.05; Test t student # P≤ 0.01; # # P≤ 0.01).

    Article Snippet: Platelet ROS-production measured by flow cytometry Production of ROS by platelets was measured by flow cytometry following the loading of platelets with the fluorescent dye, 2’ ,7’ -dichlorofluorescin diacetate (DCFH-DA) (Sigma Aldrich, UK).

    Techniques: Incubation, Flow Cytometry, Cytometry

    LPS- mediated platelet activation is associated with TXA 2 production. Human-washed platelet aggregation was performed by optical aggregometry following stimulation with U46619 (0.25μM) in the presence or absence of LPS from E . coli O111:B4 (1g/mL) after 3 min of incubation with indomethacin (10μM) or GR32191 (100ng) (A and E). The effect of U46619 (0.25μM) and LPS- induced fibrinogen binding and P-selectin exposure after incubation with indomethacin (10μM) or GR32191 (100ng) were measure in PRP by flow cytometry (B, C, F and G). Washed platelets (4 x 10 8 /mL) were pre-incubated with 10μM DCFHDA in the presence or absence of Indomethacin (10μM) or GR32191 (100ng) before being activated with U46619 (0.25μM) in the presence or absence of LPS from E . coli O111:B4 (1μg/mL) and ROS levels were analysed by flow cytometry (D and H). Cumulative data represent mean values ± SEM (n = 4). (Anova-Bonferroni test, * P≤ 0.05; ** P≤ 0.01; *** P≤ 0.001; Test t student # P≤ 0.05; # # P≤ 0.01; # # # P≤ 0.001).

    Journal: PLoS ONE

    Article Title: Lipopolysaccharide potentiates platelet responses via toll-like receptor 4-stimulated Akt-Erk-PLA2 signalling

    doi: 10.1371/journal.pone.0186981

    Figure Lengend Snippet: LPS- mediated platelet activation is associated with TXA 2 production. Human-washed platelet aggregation was performed by optical aggregometry following stimulation with U46619 (0.25μM) in the presence or absence of LPS from E . coli O111:B4 (1g/mL) after 3 min of incubation with indomethacin (10μM) or GR32191 (100ng) (A and E). The effect of U46619 (0.25μM) and LPS- induced fibrinogen binding and P-selectin exposure after incubation with indomethacin (10μM) or GR32191 (100ng) were measure in PRP by flow cytometry (B, C, F and G). Washed platelets (4 x 10 8 /mL) were pre-incubated with 10μM DCFHDA in the presence or absence of Indomethacin (10μM) or GR32191 (100ng) before being activated with U46619 (0.25μM) in the presence or absence of LPS from E . coli O111:B4 (1μg/mL) and ROS levels were analysed by flow cytometry (D and H). Cumulative data represent mean values ± SEM (n = 4). (Anova-Bonferroni test, * P≤ 0.05; ** P≤ 0.01; *** P≤ 0.001; Test t student # P≤ 0.05; # # P≤ 0.01; # # # P≤ 0.001).

    Article Snippet: Platelet ROS-production measured by flow cytometry Production of ROS by platelets was measured by flow cytometry following the loading of platelets with the fluorescent dye, 2’ ,7’ -dichlorofluorescin diacetate (DCFH-DA) (Sigma Aldrich, UK).

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

    Exposure of platelets to LPS potentiates PI3K/Akt signalling. Washed human platelets with or without treatment with U46619 (0.25μM) in the presence or absence of LPS from E . coli O111:B4 (1μg/mL) were analysed by immunoblotting using antiphospho-Akt (Ser473) antibody. Total levels of 14-3-3 ζ were measured on each sample as a loading control (A). Human-washed platelet aggregation was performed by optical aggregometry activated with U46619 (0.25μM) in the presence or absence of LPS from E . coli O111:B4 (1μg/mL) after 3 min of incubation with LY294002 (20μM), Cal (60μM) or Akt inhibitor IV (5μM) (B). The effect of U46619 and LPS- induced fibrinogen binding and P-selectin exposure after incubation with LY294002 (20μM), Cal (60μM) or Akt inhibitor IV (5μM) were measure in PRP by flow cytometry (C and D). Washed platelets (4 x 10 8 /ml) were pre-incubated with 10μM DCFH-DA in the presence or absence of LY294002 (20μM), Cal (60μM) or Akt inhibitor IV (5μM) before being activated with U46619 (0.25μM) in the presence or absence of LPS from E . coli O111:B4 (1μg/mL) and ROS levels were analysed by flow cytometry (E). Cumulative data represent mean values ± SEM (n = 4). (Anova-Bonferroni test, * P≤ 0.05; ** P≤ 0.01; *** P≤ 0.001; Test t student # # P≤ 0.01).

    Journal: PLoS ONE

    Article Title: Lipopolysaccharide potentiates platelet responses via toll-like receptor 4-stimulated Akt-Erk-PLA2 signalling

    doi: 10.1371/journal.pone.0186981

    Figure Lengend Snippet: Exposure of platelets to LPS potentiates PI3K/Akt signalling. Washed human platelets with or without treatment with U46619 (0.25μM) in the presence or absence of LPS from E . coli O111:B4 (1μg/mL) were analysed by immunoblotting using antiphospho-Akt (Ser473) antibody. Total levels of 14-3-3 ζ were measured on each sample as a loading control (A). Human-washed platelet aggregation was performed by optical aggregometry activated with U46619 (0.25μM) in the presence or absence of LPS from E . coli O111:B4 (1μg/mL) after 3 min of incubation with LY294002 (20μM), Cal (60μM) or Akt inhibitor IV (5μM) (B). The effect of U46619 and LPS- induced fibrinogen binding and P-selectin exposure after incubation with LY294002 (20μM), Cal (60μM) or Akt inhibitor IV (5μM) were measure in PRP by flow cytometry (C and D). Washed platelets (4 x 10 8 /ml) were pre-incubated with 10μM DCFH-DA in the presence or absence of LY294002 (20μM), Cal (60μM) or Akt inhibitor IV (5μM) before being activated with U46619 (0.25μM) in the presence or absence of LPS from E . coli O111:B4 (1μg/mL) and ROS levels were analysed by flow cytometry (E). Cumulative data represent mean values ± SEM (n = 4). (Anova-Bonferroni test, * P≤ 0.05; ** P≤ 0.01; *** P≤ 0.001; Test t student # # P≤ 0.01).

    Article Snippet: Platelet ROS-production measured by flow cytometry Production of ROS by platelets was measured by flow cytometry following the loading of platelets with the fluorescent dye, 2’ ,7’ -dichlorofluorescin diacetate (DCFH-DA) (Sigma Aldrich, UK).

    Techniques: Incubation, Binding Assay, Flow Cytometry, Cytometry

    LPS potenciates U46619-stimulated platelet activation. Human-washed platelet aggregation was performed by optical aggregometry following stimulation with U46619 (0.25μM) or CRP-XL (0.25μg/ml) in the presence or absence of LPS from E . coli O111:B4 (0.5, 1, 5 or 7.5μg/mL) or LPS RS (1 or 7.5 μg/mL) (A, B, E, F). The effect of U46619 or CRP-XL and LPS- on fibrinogen binding and P-selectin exposure was measured in PRP by flow cytometry (C, D, G, H). Cumulative data represent mean values ± SEM (n = 4). (Anova-Bonferroni test, * P≤ 0.05; ** P≤ 0.01; *** P≤ 0.001).

    Journal: PLoS ONE

    Article Title: Lipopolysaccharide potentiates platelet responses via toll-like receptor 4-stimulated Akt-Erk-PLA2 signalling

    doi: 10.1371/journal.pone.0186981

    Figure Lengend Snippet: LPS potenciates U46619-stimulated platelet activation. Human-washed platelet aggregation was performed by optical aggregometry following stimulation with U46619 (0.25μM) or CRP-XL (0.25μg/ml) in the presence or absence of LPS from E . coli O111:B4 (0.5, 1, 5 or 7.5μg/mL) or LPS RS (1 or 7.5 μg/mL) (A, B, E, F). The effect of U46619 or CRP-XL and LPS- on fibrinogen binding and P-selectin exposure was measured in PRP by flow cytometry (C, D, G, H). Cumulative data represent mean values ± SEM (n = 4). (Anova-Bonferroni test, * P≤ 0.05; ** P≤ 0.01; *** P≤ 0.001).

    Article Snippet: Platelet ROS-production measured by flow cytometry Production of ROS by platelets was measured by flow cytometry following the loading of platelets with the fluorescent dye, 2’ ,7’ -dichlorofluorescin diacetate (DCFH-DA) (Sigma Aldrich, UK).

    Techniques: Activation Assay, Binding Assay, Flow Cytometry, Cytometry

    Activation of NPM1 expression is highly associated with EBV infection. A). The immunoblots for NPM1, EBNA2, c-MYC, and actin control from EBV latently infected type I AKATA BL cells, type II HONE1 nasopharyngeal carcinoma cells (NPC), type III LCLs, and EBV negatively infected AKATA, NPC HONE1, and primary B cells. B). The mRNA expression levels of NPM1 in primary B cells and four individual LCLs were determined by quantitative real-time PCR (qPCR), respectively. The amount of NPM1 mRNA is expressed in relation to that of the GAPDH control. C). The effects of EBNA2 and EBNALP on the NPM1-Luc reporter plasmid were determined by the transfection-mediated reporter assay. Error bars represent the standard deviation from the mean for data from at least three independent experiments in this and subsequent figures. The immunoblotting images for transfected EBNA2, EBNALP, and the actin control are shown. D). The expression levels of NPM1, EBNA2, and actin in primary B cells, BJAB, or BJAB-EBNA2 were determined. E–F). Primary B cells (5×10 4 ) were infected with EBV or PBS (Mock) and subjected to an IF staining protocol using antibodies for EBNA2 (v-C20), NPM1 (C-19), or c-MYC at 0, 3 or 7 days after infection (dai) followed by a donkey anti-goat antibody conjugated to FITC (Green) or a goat anti-mouse antibody conjugated to rhodamine (red). For mock infection, the Y-axis represented the signaling resulted from a double staining procedure using c-MYC and NPM1 antibodies, whereas the X-axis represented the signaling resulted from an EBNA2 immunostaining protocol. Nuclei were counterstained with DAPI. The immunostained cells were visualized by fluorescent microscopy and quantified by flow cytometry (F). G). The newly established LCL (LCL-New), or previously established LCL (LCL1) and primary B cells were assayed for EBNA2, c-MYC, and actin control expression levels.

    Journal: PLoS Pathogens

    Article Title: The Nuclear Chaperone Nucleophosmin Escorts an Epstein-Barr Virus Nuclear Antigen to Establish Transcriptional Cascades for Latent Infection in Human B Cells

    doi: 10.1371/journal.ppat.1003084

    Figure Lengend Snippet: Activation of NPM1 expression is highly associated with EBV infection. A). The immunoblots for NPM1, EBNA2, c-MYC, and actin control from EBV latently infected type I AKATA BL cells, type II HONE1 nasopharyngeal carcinoma cells (NPC), type III LCLs, and EBV negatively infected AKATA, NPC HONE1, and primary B cells. B). The mRNA expression levels of NPM1 in primary B cells and four individual LCLs were determined by quantitative real-time PCR (qPCR), respectively. The amount of NPM1 mRNA is expressed in relation to that of the GAPDH control. C). The effects of EBNA2 and EBNALP on the NPM1-Luc reporter plasmid were determined by the transfection-mediated reporter assay. Error bars represent the standard deviation from the mean for data from at least three independent experiments in this and subsequent figures. The immunoblotting images for transfected EBNA2, EBNALP, and the actin control are shown. D). The expression levels of NPM1, EBNA2, and actin in primary B cells, BJAB, or BJAB-EBNA2 were determined. E–F). Primary B cells (5×10 4 ) were infected with EBV or PBS (Mock) and subjected to an IF staining protocol using antibodies for EBNA2 (v-C20), NPM1 (C-19), or c-MYC at 0, 3 or 7 days after infection (dai) followed by a donkey anti-goat antibody conjugated to FITC (Green) or a goat anti-mouse antibody conjugated to rhodamine (red). For mock infection, the Y-axis represented the signaling resulted from a double staining procedure using c-MYC and NPM1 antibodies, whereas the X-axis represented the signaling resulted from an EBNA2 immunostaining protocol. Nuclei were counterstained with DAPI. The immunostained cells were visualized by fluorescent microscopy and quantified by flow cytometry (F). G). The newly established LCL (LCL-New), or previously established LCL (LCL1) and primary B cells were assayed for EBNA2, c-MYC, and actin control expression levels.

    Article Snippet: The immunostained cells were visualized using the Cell Imaging Station (Life Technologies) or analyzed by Guava flow cytometry (Millipore).

    Techniques: Activation Assay, Expressing, Infection, Western Blot, Real-time Polymerase Chain Reaction, Plasmid Preparation, Transfection, Reporter Assay, Standard Deviation, Staining, Double Staining, Immunostaining, Microscopy, Flow Cytometry, Cytometry

    Increased cell cycle arrest in irradiated parotid glands pretreated with Roscovitine. The head and neck region of female FVB mice was treated with a 5Gy dose of radiation with or without 100 mg/kg Roscovitine pretreatment. Parotid glands were removed 6 hours after radiation treatment. A) Representative flow cytometry histograms from untreated (UT), irradiated with vehicle pre-treatment (IR+V), Roscovitine alone (Rosco), and Roscovitine prior to irradiation (IR+Rosco). B) Tissues were dispersed, stained with propidium iodide, and analyzed by flow cytometry. Graphical representation of the mean percentage of cells gated in G2/M with SEM from ≥9 mice per treatment. C) RNA was isolated from parotid glands, treated as stated above and real-time RT-PCR was run with primers for p21 amplification. Results were calculated using the 2 −ΔΔCt , normalized to untreated and displayed as mean with SEM≥4 mice per treatment. D) Parotid tissues were treated as stated above and collected 6 hours following radiation for immunoblotting and prepared as described in the materials and methods, and membranes were probed for p21 (top panel) and cdc2 Tyr15 (middle panel), with total ERK (bottom panel) as a loading control. Tissues were collected at 24 hours (E) and 48 hours (F) from irradiated mice pretreated with Roscovitine, embedded in paraffin, and stained for PCNA as described in the materials and methods. Results are graphed as the number of PCNA-positive acinar cells as a percentage of total counted acinar cells. The data are shown as the mean with SEM≥3 mice per treatment group. Treatment groups with the same letters are not statistically different from each other.

    Journal: PLoS ONE

    Article Title: Prevention of Radiation-Induced Salivary Gland Dysfunction Utilizing a CDK Inhibitor in a Mouse Model

    doi: 10.1371/journal.pone.0051363

    Figure Lengend Snippet: Increased cell cycle arrest in irradiated parotid glands pretreated with Roscovitine. The head and neck region of female FVB mice was treated with a 5Gy dose of radiation with or without 100 mg/kg Roscovitine pretreatment. Parotid glands were removed 6 hours after radiation treatment. A) Representative flow cytometry histograms from untreated (UT), irradiated with vehicle pre-treatment (IR+V), Roscovitine alone (Rosco), and Roscovitine prior to irradiation (IR+Rosco). B) Tissues were dispersed, stained with propidium iodide, and analyzed by flow cytometry. Graphical representation of the mean percentage of cells gated in G2/M with SEM from ≥9 mice per treatment. C) RNA was isolated from parotid glands, treated as stated above and real-time RT-PCR was run with primers for p21 amplification. Results were calculated using the 2 −ΔΔCt , normalized to untreated and displayed as mean with SEM≥4 mice per treatment. D) Parotid tissues were treated as stated above and collected 6 hours following radiation for immunoblotting and prepared as described in the materials and methods, and membranes were probed for p21 (top panel) and cdc2 Tyr15 (middle panel), with total ERK (bottom panel) as a loading control. Tissues were collected at 24 hours (E) and 48 hours (F) from irradiated mice pretreated with Roscovitine, embedded in paraffin, and stained for PCNA as described in the materials and methods. Results are graphed as the number of PCNA-positive acinar cells as a percentage of total counted acinar cells. The data are shown as the mean with SEM≥3 mice per treatment group. Treatment groups with the same letters are not statistically different from each other.

    Article Snippet: Flow Cytometry Pairs of parotid glands were minced in dispersion media of 1 mg/ml Hyaluronidase (Sigma-Aldrich, St. Louis, MO), 1 mg/ml Collagenase P (Roche Diagnostics, Indianapolis, IN), in Modified Hanks Solution mixed at 37°C for 20 minutes, re-suspended with 1µM EGTA (Fisher Scientific), and further mixed at 37°C for 10 minutes.

    Techniques: Irradiation, Mouse Assay, Flow Cytometry, Cytometry, Staining, Isolation, Quantitative RT-PCR, Amplification

    Selectivity effect of plasma treatment: B16 melanoma cells treated with the cold plasma device for 0, 30, 60 and 120 s. ( A ) 24 h; ( B ) 48 h. Annexin V and 7-AAD staining was performed for flow cytometry analysis at 24 and 48 h after treatment. Four-quadrant analysis of the results characterises the cells as viable (unstained), apoptotic (Annexin V positive), late-apoptotic (double positive) and dead (7-AAD positive).

    Journal: British Journal of Cancer

    Article Title: Cold plasma selectivity and the possibility of a paradigm shift in cancer therapy

    doi: 10.1038/bjc.2011.386

    Figure Lengend Snippet: Selectivity effect of plasma treatment: B16 melanoma cells treated with the cold plasma device for 0, 30, 60 and 120 s. ( A ) 24 h; ( B ) 48 h. Annexin V and 7-AAD staining was performed for flow cytometry analysis at 24 and 48 h after treatment. Four-quadrant analysis of the results characterises the cells as viable (unstained), apoptotic (Annexin V positive), late-apoptotic (double positive) and dead (7-AAD positive).

    Article Snippet: Flow Cytometry Triplicate samples of 1 × 104 murine macrophages and B16-F10 melanoma cells were plated into 96-well plates with 100 μ l of D10 media (DMEM (Sigma-Aldrich, St Louis, MO, USA)), supplemented with 10% fetal bovine serum, penicillin (100 IU ml−1 ), streptomycin (100 μ g ml−1 ; Sigma-Aldrich) and 1% -Glutamine (Mediatech Inc., Manassas, VA, USA)) per well.

    Techniques: Staining, Flow Cytometry, Cytometry

    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

    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

    Examples of flow cytometry plots of side scatter (SS) versus green fluorescence of different enrichment populations. Cells were stained with SYTO 13. Arrows show the position of 1-μm beads. (A) Melted ice sample; (B) round II filtered R2B culture

    Journal:

    Article Title: Detection and Isolation of Ultrasmall Microorganisms from a 120,000-Year-Old Greenland Glacier Ice Core

    doi: 10.1128/AEM.71.12.7806-7818.2005

    Figure Lengend Snippet: Examples of flow cytometry plots of side scatter (SS) versus green fluorescence of different enrichment populations. Cells were stained with SYTO 13. Arrows show the position of 1-μm beads. (A) Melted ice sample; (B) round II filtered R2B culture

    Article Snippet: In addition to careful setting of the flow cytometry parameters to avoid the instrument noise, we used autoclaved H2 O that had been filtered through a 0.1-μm Millipore filter as a sheath solution, filtered dimethyl sulfoxide for dilution of SYTO13, a small quantity of beads added to samples with low cell density, and extended times (20,000 events or 300 s) for each analysis at a low rate.

    Techniques: Flow Cytometry, Cytometry, Fluorescence, Staining

    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

    Relative quantification assays for colistin-induced ROS levels in E. coli expressing mcr -like genetic determinant. a Confocal microscopy-based quantification of ROS levels in E. coli carrying different versions of MCR family. b Use of flow cytometry to determine the ROS fluctuation in E. coli having mcr variants. The ratio of fluorescent cells ( a ) was calculated by counting the number of cells with/without fluorescence (seen in Supplementary Figure 10 ). Over 500 cells from 6 individual photographs were counted for each group. The data was given after one-way analysis of variance (ANOVA) along with Tukey–Kramer multiple comparisons post hoc test 36 . Statistical significance was set at p

    Journal: Communications Biology

    Article Title: Action and mechanism of the colistin resistance enzyme MCR-4

    doi: 10.1038/s42003-018-0278-1

    Figure Lengend Snippet: Relative quantification assays for colistin-induced ROS levels in E. coli expressing mcr -like genetic determinant. a Confocal microscopy-based quantification of ROS levels in E. coli carrying different versions of MCR family. b Use of flow cytometry to determine the ROS fluctuation in E. coli having mcr variants. The ratio of fluorescent cells ( a ) was calculated by counting the number of cells with/without fluorescence (seen in Supplementary Figure 10 ). Over 500 cells from 6 individual photographs were counted for each group. The data was given after one-way analysis of variance (ANOVA) along with Tukey–Kramer multiple comparisons post hoc test 36 . Statistical significance was set at p

    Article Snippet: Flow cytometry-based measurement of the intracellular ROS level A mid-log phase culture of E. coli (OD600, 0.5) was utilized to measure the intracellular level of ROS, in which 10 mM of oxidant sensor dye DCFH2-DA (Sigma) was supplemented and maintained for 0.5 h. When necessary, colistin was added.

    Techniques: Expressing, Confocal Microscopy, Flow Cytometry, Cytometry, Fluorescence

    In vivo contribution of B-CPC to homeostasis in the adult mammalian heart. a Timeline for B-CPC and progeny analysis. b Immunohistochemistry of mature YFP + SαA-positive CM surrounded by laminin, at 6 ( n = 2) and 12 months ( n = 2) post-TM induction, were scattered throughout the heart. Bars, 20 μm (left), 50 μm (right). c - f Flow cytometry of total adult CM from ( c ) Bmi1 -YFP NI ( n = 3), ( d ) Bmi1 -YFP 5d-postTM ( n = 8), ( e ) Bmi1 -YFP at two months ( n = 5) and ( f ) Bmi1 -YFP mice one year post-TM induction (n = 6). g Expression of contractility proteins in YFP + and YFP − CM; SαA, tropomyosin (TPM) and connexin 43 (CX43). Bars, 50 μm. h Number of YFP + CM, measured by flow cytometry and immunocytochemistry, increases from 2 to 12 months post-TM induction, 3 ± 0.6 % at 2 months (2m; n = 7), 3.8 ± 0.7 % at 6 months (6m; n = 3) and 6.7 ± 2 % at 12 months (12m; n = 9). Data shown as mean ± SEM. P value was calculated by unpaired Student’s t-test with Welch’s correction. * P

    Journal: Stem Cell Research & Therapy

    Article Title: Cardiac Bmi1+ cells contribute to myocardial renewal in the murine adult heart

    doi: 10.1186/s13287-015-0196-9

    Figure Lengend Snippet: In vivo contribution of B-CPC to homeostasis in the adult mammalian heart. a Timeline for B-CPC and progeny analysis. b Immunohistochemistry of mature YFP + SαA-positive CM surrounded by laminin, at 6 ( n = 2) and 12 months ( n = 2) post-TM induction, were scattered throughout the heart. Bars, 20 μm (left), 50 μm (right). c - f Flow cytometry of total adult CM from ( c ) Bmi1 -YFP NI ( n = 3), ( d ) Bmi1 -YFP 5d-postTM ( n = 8), ( e ) Bmi1 -YFP at two months ( n = 5) and ( f ) Bmi1 -YFP mice one year post-TM induction (n = 6). g Expression of contractility proteins in YFP + and YFP − CM; SαA, tropomyosin (TPM) and connexin 43 (CX43). Bars, 50 μm. h Number of YFP + CM, measured by flow cytometry and immunocytochemistry, increases from 2 to 12 months post-TM induction, 3 ± 0.6 % at 2 months (2m; n = 7), 3.8 ± 0.7 % at 6 months (6m; n = 3) and 6.7 ± 2 % at 12 months (12m; n = 9). Data shown as mean ± SEM. P value was calculated by unpaired Student’s t-test with Welch’s correction. * P

    Article Snippet: Cell isolation, culture and flow cytometry Hearts were collected from Bmi1 -YFP mice five days after TM induction, perfused with PBS to remove blood cells, and processed by enzymatic digestion using 0.1 % collagenase IV (Sigma) and 10 μg/ml DNAse (Roche, Madrid, Spain) (40 min, 37 °C).

    Techniques: In Vivo, Immunohistochemistry, Flow Cytometry, Cytometry, Mouse Assay, Expressing, Immunocytochemistry

    A subset of humans harbor microbiota with superantigen-type binding to monoclonal antibodies expressing murine VH5/6/7 and human VH3 variable regions. (A) Bacterial flow cytometry analysis indicating the percent of fecal bacteria from ten human donors grafted into germ-free mice that were coated with endogenous polyclonal IgA (open squares), or various negative control B2 mAbs (open circles) or microbiota-reactive SI IgA mAbs (closed circles) expressed with a human IgG1 backbone and detected with anti-hIgG reagents. Each circle represents a distinct mAb. Red squares highlight the four individuals with superantigen-type reactivity. Asterisks denote microbiota from cow’s milk allergic individuals, and the remaining samples were from healthy individuals. Data compiled from four independent experiments. (B) Bacterial flow cytometry analysis of the four human individuals’ microbiota from panel 1A that showed superantigen-type reactivity with a larger panel of 53 murine SI IgA-derived (closed circles) or 47 naïve B2-derived mAbs (open circles), grouped by heavy chain variable gene usage as indicated. (C) Representative flow cytometry plots and (D) Summary of bacterial flow cytometry analysis of indicated strains cultured in vitro and stained with 53 SI IgA or 47 B2-derived mAbs, grouped by heavy chain variable gene usage as indicated. Data compiled from three independent experiments. (E) Representative flow cytometry plots and (F) Summary bacterial flow cytometry analysis of indicated strains cultured in vitro and stained with 26 fully human anti-influenza mAbs, grouped by heavy chain variable gene usage as indicated. Data compiled from three independent experiments. P values in panels (B-F) calculated by unpaired t test.

    Journal: Science translational medicine

    Article Title: B cell superantigens in the human intestinal microbiota

    doi: 10.1126/scitranslmed.aau9356

    Figure Lengend Snippet: A subset of humans harbor microbiota with superantigen-type binding to monoclonal antibodies expressing murine VH5/6/7 and human VH3 variable regions. (A) Bacterial flow cytometry analysis indicating the percent of fecal bacteria from ten human donors grafted into germ-free mice that were coated with endogenous polyclonal IgA (open squares), or various negative control B2 mAbs (open circles) or microbiota-reactive SI IgA mAbs (closed circles) expressed with a human IgG1 backbone and detected with anti-hIgG reagents. Each circle represents a distinct mAb. Red squares highlight the four individuals with superantigen-type reactivity. Asterisks denote microbiota from cow’s milk allergic individuals, and the remaining samples were from healthy individuals. Data compiled from four independent experiments. (B) Bacterial flow cytometry analysis of the four human individuals’ microbiota from panel 1A that showed superantigen-type reactivity with a larger panel of 53 murine SI IgA-derived (closed circles) or 47 naïve B2-derived mAbs (open circles), grouped by heavy chain variable gene usage as indicated. (C) Representative flow cytometry plots and (D) Summary of bacterial flow cytometry analysis of indicated strains cultured in vitro and stained with 53 SI IgA or 47 B2-derived mAbs, grouped by heavy chain variable gene usage as indicated. Data compiled from three independent experiments. (E) Representative flow cytometry plots and (F) Summary bacterial flow cytometry analysis of indicated strains cultured in vitro and stained with 26 fully human anti-influenza mAbs, grouped by heavy chain variable gene usage as indicated. Data compiled from three independent experiments. P values in panels (B-F) calculated by unpaired t test.

    Article Snippet: In order to avoid cross reactions with monoclonal antibodies used for flow cytometry stainings, in vitro stimulated mouse and human B cells were incubated for 30 min at 4–8°C with high dose human IgG (1mg/ml; Sigma) prior to the addition of fluorophore-conjugated antibodies to block all binding sites of Ibps potentially still bound to the stimulated cells.

    Techniques: Binding Assay, Expressing, Flow Cytometry, Mouse Assay, Negative Control, Derivative Assay, Cell Culture, In Vitro, Staining

    IbpA and IbpB activate murine and human B cells in vitro. (A) Representative flow cytometry analyses and summary plots depicting purified murine B cells from wild-type or Myd88 −/− mice 6 hours after in vitro incubation with indicated stimuli and analyzed for surface expression of CD19 and IgM or (B) CD69. Data pooled from two independent experiments. (C) Representative flow cytometry analyses and summary plots depicting purified human B cells 6 hours after in vitro incubation with indicated stimuli and analyzed for surface expression of CD19 and IgM or (D) CD83 and CD69. Each data point represents one human sample. Samples were obtained from three individuals and cells from one individual were isolated and analyzed in two independent experiments. Data pooled from two independent experiments. P values calculated by unpaired t test.

    Journal: Science translational medicine

    Article Title: B cell superantigens in the human intestinal microbiota

    doi: 10.1126/scitranslmed.aau9356

    Figure Lengend Snippet: IbpA and IbpB activate murine and human B cells in vitro. (A) Representative flow cytometry analyses and summary plots depicting purified murine B cells from wild-type or Myd88 −/− mice 6 hours after in vitro incubation with indicated stimuli and analyzed for surface expression of CD19 and IgM or (B) CD69. Data pooled from two independent experiments. (C) Representative flow cytometry analyses and summary plots depicting purified human B cells 6 hours after in vitro incubation with indicated stimuli and analyzed for surface expression of CD19 and IgM or (D) CD83 and CD69. Each data point represents one human sample. Samples were obtained from three individuals and cells from one individual were isolated and analyzed in two independent experiments. Data pooled from two independent experiments. P values calculated by unpaired t test.

    Article Snippet: In order to avoid cross reactions with monoclonal antibodies used for flow cytometry stainings, in vitro stimulated mouse and human B cells were incubated for 30 min at 4–8°C with high dose human IgG (1mg/ml; Sigma) prior to the addition of fluorophore-conjugated antibodies to block all binding sites of Ibps potentially still bound to the stimulated cells.

    Techniques: In Vitro, Flow Cytometry, Purification, Mouse Assay, Incubation, Expressing, Isolation

    Superantigen-expressing strains stimulate and attract IgA in vivo. (A) Representative flow cytometry plots gated on FSC hi Lineage- cells or (B) absolute number summaries of SI LP IgA PCs from GF or gnotobiotic mice four weeks after monocolonization with indicated bacterial strains. Data compiled from three independent experiments. (C) Absolute number of SI IgA PCs expressing indicated variable gene families. Calculated from the total cell number shown in panel (B) multiplied by the percent of the repertoire expressing indicated variable region genes as determined by DNA sequencing. One-way ANOVA p values were 0.0048 for VH5/6/7, 0.5156 for VH1–47, and 0.6958 for VH3–5. P values in panels (B-C) calculated by unpaired t test.

    Journal: Science translational medicine

    Article Title: B cell superantigens in the human intestinal microbiota

    doi: 10.1126/scitranslmed.aau9356

    Figure Lengend Snippet: Superantigen-expressing strains stimulate and attract IgA in vivo. (A) Representative flow cytometry plots gated on FSC hi Lineage- cells or (B) absolute number summaries of SI LP IgA PCs from GF or gnotobiotic mice four weeks after monocolonization with indicated bacterial strains. Data compiled from three independent experiments. (C) Absolute number of SI IgA PCs expressing indicated variable gene families. Calculated from the total cell number shown in panel (B) multiplied by the percent of the repertoire expressing indicated variable region genes as determined by DNA sequencing. One-way ANOVA p values were 0.0048 for VH5/6/7, 0.5156 for VH1–47, and 0.6958 for VH3–5. P values in panels (B-C) calculated by unpaired t test.

    Article Snippet: In order to avoid cross reactions with monoclonal antibodies used for flow cytometry stainings, in vitro stimulated mouse and human B cells were incubated for 30 min at 4–8°C with high dose human IgG (1mg/ml; Sigma) prior to the addition of fluorophore-conjugated antibodies to block all binding sites of Ibps potentially still bound to the stimulated cells.

    Techniques: Expressing, In Vivo, Flow Cytometry, Mouse Assay, DNA Sequencing

    Growth curves of DENV2 in monocytes of G6PD-deficient and normal controls. The monocytes from G6PD-deficient and normal controls were infected with DENV2 at an MOI of 0.1. Cells and culture supernatants were harvested at 24, 48, 72, 96, 120 hours post-infection. Number of DENV infected cells were assayed by flow cytometry ( A ), whereas virus released by the infected cells was determined by Plaque assay ( B ). Number of infected cells as well as virus titer found to be significantly higher in infected monocytes from G6PD-deficient individuals compared to the normal controls.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Dengue Virus Type 2 (DENV2)-Induced Oxidative Responses in Monocytes from Glucose-6-Phosphate Dehydrogenase (G6PD)-Deficient and G6PD Normal Subjects

    doi: 10.1371/journal.pntd.0002711

    Figure Lengend Snippet: Growth curves of DENV2 in monocytes of G6PD-deficient and normal controls. The monocytes from G6PD-deficient and normal controls were infected with DENV2 at an MOI of 0.1. Cells and culture supernatants were harvested at 24, 48, 72, 96, 120 hours post-infection. Number of DENV infected cells were assayed by flow cytometry ( A ), whereas virus released by the infected cells was determined by Plaque assay ( B ). Number of infected cells as well as virus titer found to be significantly higher in infected monocytes from G6PD-deficient individuals compared to the normal controls.

    Article Snippet: Intracellular detection of DENV2 by flow cytometry Infected and mock-infected monocytes were harvested and washed twice with cold phosphate buffered saline (PBS) containing 0.1% NaN3 (SIGMA-USA).

    Techniques: Infection, Flow Cytometry, Cytometry, Plaque Assay

    Engagement of CD160 by HLA-C, constitutively expressed by K562, triggers NK cell cytotoxicity. ( A ) K562 cells were analyzed by flow cytometry for surface expression of HLA class I using broad anti-HLA-class I (mAb W6/32), anti-HLA-C (mAb B1.23.2), anti-HLA-C free heavy chains (mAb L31), anti-HLA-E (mAb MEM-E/06), or anti-HLA-G (mAb 87G) (black profiles) followed by PE-labeled conjugate. Open profiles are Ig-isotype control stainings. Data are representative of several independent experiments. ( B ) Cytotoxicity of NK92 cultured without IL-2 against K562 in the absence of mAb (no mAb) or in the presence of mAb MEM-E/06 (HLA-E, 5 μg/ml), mAb B1.23.2 (HLA-C, 10 μg/ml), or Ig-isotype control (IgG). Results are mean ± SD of triplicates and are representative of several independent experiments.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Engagement of CD160 receptor by HLA-C is a triggering mechanism used by circulating natural killer (NK) cells to mediate cytotoxicity

    doi: 10.1073/pnas.012681099

    Figure Lengend Snippet: Engagement of CD160 by HLA-C, constitutively expressed by K562, triggers NK cell cytotoxicity. ( A ) K562 cells were analyzed by flow cytometry for surface expression of HLA class I using broad anti-HLA-class I (mAb W6/32), anti-HLA-C (mAb B1.23.2), anti-HLA-C free heavy chains (mAb L31), anti-HLA-E (mAb MEM-E/06), or anti-HLA-G (mAb 87G) (black profiles) followed by PE-labeled conjugate. Open profiles are Ig-isotype control stainings. Data are representative of several independent experiments. ( B ) Cytotoxicity of NK92 cultured without IL-2 against K562 in the absence of mAb (no mAb) or in the presence of mAb MEM-E/06 (HLA-E, 5 μg/ml), mAb B1.23.2 (HLA-C, 10 μg/ml), or Ig-isotype control (IgG). Results are mean ± SD of triplicates and are representative of several independent experiments.

    Article Snippet: Flow cytometry binding experiments were performed using culture supernatant from a soluble CD160 transfectant cultured in serum-free medium, followed by incubation with anti-Flag mAb (Sigma) and PE-labeled conjugate.

    Techniques: Flow Cytometry, Cytometry, Expressing, Labeling, Cell Culture

    Activation of fresh peripheral blood NK cell-mediated cytolysis by CD160. ( A ) Cytotoxic activity of freshly isolated human PB-NK separated in CD160 + and -160 − populations against K562 target cell in the absence of mAb (no mAb) or in the presence of mAb CL1-R2 (CD160). ( B ) Cytotoxic activity of PB-NK either untreated (CD160 bright+ ) or cultured for 3 days with IL-2 (CD160 dim+ ) against K562 target cell in the absence of mAb (no mAb) or in the presence of mAb CL1-R2 or Ig-isotype control (IgG1). ( C ) CD160 expression in fresh PB-NK cells either untreated (−IL-2) or cultured for 3 days with IL-2 (+IL-2) was analyzed by flow cytometry using FITC-labeled BY55 mAb. Data are representative of several independent experiments. Open profile is Ig-isotype control staining. ( D ) Redirected lysis assay of P815 using fresh human CD160 + PB-NK in the presence of mAbs specific for the indicated molecules or control IgG1. The effector-to-target cell (E:T) ratio used was 25. Results of A , B , and D are mean ± SD of triplicates and are representative of three independent experiments.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Engagement of CD160 receptor by HLA-C is a triggering mechanism used by circulating natural killer (NK) cells to mediate cytotoxicity

    doi: 10.1073/pnas.012681099

    Figure Lengend Snippet: Activation of fresh peripheral blood NK cell-mediated cytolysis by CD160. ( A ) Cytotoxic activity of freshly isolated human PB-NK separated in CD160 + and -160 − populations against K562 target cell in the absence of mAb (no mAb) or in the presence of mAb CL1-R2 (CD160). ( B ) Cytotoxic activity of PB-NK either untreated (CD160 bright+ ) or cultured for 3 days with IL-2 (CD160 dim+ ) against K562 target cell in the absence of mAb (no mAb) or in the presence of mAb CL1-R2 or Ig-isotype control (IgG1). ( C ) CD160 expression in fresh PB-NK cells either untreated (−IL-2) or cultured for 3 days with IL-2 (+IL-2) was analyzed by flow cytometry using FITC-labeled BY55 mAb. Data are representative of several independent experiments. Open profile is Ig-isotype control staining. ( D ) Redirected lysis assay of P815 using fresh human CD160 + PB-NK in the presence of mAbs specific for the indicated molecules or control IgG1. The effector-to-target cell (E:T) ratio used was 25. Results of A , B , and D are mean ± SD of triplicates and are representative of three independent experiments.

    Article Snippet: Flow cytometry binding experiments were performed using culture supernatant from a soluble CD160 transfectant cultured in serum-free medium, followed by incubation with anti-Flag mAb (Sigma) and PE-labeled conjugate.

    Techniques: Activation Assay, Activity Assay, Isolation, Cell Culture, Expressing, Flow Cytometry, Cytometry, Labeling, Staining, Lysis

    Soluble recombinant HLA-Cw3 and CD160 proteins bind to JAR-Cw3 and Jurkat-CD160, respectively. ( A Upper ) By flow cytometry, mAb CL1-R2 stained Jurkat-CD160 but not Jurkat cells (black profiles). Open profiles are Ig-isotype control stainings. ( A Lower ) flow cytometry binding of soluble HLA-Cw3 cross-linked with B1.23.2 mAb, followed by incubation with PE-streptavidin on Jurkat-CD160 and NK.3.3 cells but not Jurkat (black profiles). Open profiles are control staining with B1.23.2 mAb and streptavidin-PE. ( B ) Flow cytometry showing binding of mAb B1.23.2 on JAR-Cw3 but not JAR ( Upper , open profiles are Ig-isotype control staining), and of soluble CD160-Flag, after incubation with anti-Flag mAb followed by PE-labeled conjugate on Jurkat-CD160 and NK.3.3 cells but not Jurkat ( Lower , open profiles are staining after incubation with control Jurkat supernatant). ( C ) Cytotoxicity of NK92 cultured without IL-2 against JAR-Cw3 target cells in the absence of mAb (no mAb) or in the presence of mAb CL1-R2 (CD160). Results are mean ± SD of triplicates and are representative of three independent experiments.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Engagement of CD160 receptor by HLA-C is a triggering mechanism used by circulating natural killer (NK) cells to mediate cytotoxicity

    doi: 10.1073/pnas.012681099

    Figure Lengend Snippet: Soluble recombinant HLA-Cw3 and CD160 proteins bind to JAR-Cw3 and Jurkat-CD160, respectively. ( A Upper ) By flow cytometry, mAb CL1-R2 stained Jurkat-CD160 but not Jurkat cells (black profiles). Open profiles are Ig-isotype control stainings. ( A Lower ) flow cytometry binding of soluble HLA-Cw3 cross-linked with B1.23.2 mAb, followed by incubation with PE-streptavidin on Jurkat-CD160 and NK.3.3 cells but not Jurkat (black profiles). Open profiles are control staining with B1.23.2 mAb and streptavidin-PE. ( B ) Flow cytometry showing binding of mAb B1.23.2 on JAR-Cw3 but not JAR ( Upper , open profiles are Ig-isotype control staining), and of soluble CD160-Flag, after incubation with anti-Flag mAb followed by PE-labeled conjugate on Jurkat-CD160 and NK.3.3 cells but not Jurkat ( Lower , open profiles are staining after incubation with control Jurkat supernatant). ( C ) Cytotoxicity of NK92 cultured without IL-2 against JAR-Cw3 target cells in the absence of mAb (no mAb) or in the presence of mAb CL1-R2 (CD160). Results are mean ± SD of triplicates and are representative of three independent experiments.

    Article Snippet: Flow cytometry binding experiments were performed using culture supernatant from a soluble CD160 transfectant cultured in serum-free medium, followed by incubation with anti-Flag mAb (Sigma) and PE-labeled conjugate.

    Techniques: Recombinant, Flow Cytometry, Cytometry, Staining, Binding Assay, Incubation, Labeling, Cell Culture

    Abortive infection of T-cells results in ER-stress induced autophagy. ( A ) Flow cytometry analysis of intracellular Ca 2+ levels in primary CD4 + T-cells exposed to ionomycin, CD3/CD28 beads or EBOV VLPs for 10 min. Top dot plots results represent bound Ca 2+ (X axis) and free Ca 2+ (Y axis). Bottom dot plots represent the ratio of free Ca 2+ /Bound Ca 2+ over time during the 10 min stimulation. ( B ) Top: Western blot analysis of isolated CD4 + T-cells exposed to EBOV at various MOIs or mock-infected cells (M) for markers of ER-stress phospho-IRE1a and XBP1, as well as dephosphorylated form of IRE1a. Bottom: Western blot analysis of isolated CD4 + T-cells exposed to purified EBOV for XBP1. ( C ) Western blot analysis of phosphorylated eIF2a (P-eIF2a) in CD4 + T-cells exposed to EBOV in the presence or absence 1E7-03, okadaic acid (OA), a Lck inhibitor PP2 or a PI3K inhibitor Ly294. The ratio of phospho-eIF2a to tubulin is indicated below the Western blot images. ( D ) The induction of autophagy assessed by staining for LC3 in CD4 + T-cells exposed to EBOV or treated with rapamycin. Left: representative primary flow cytometry data. The gate indicates the position of LC3-positive cells based on the lack of staining with isotype control antibodies. Right: mean values based on triplicate samples from two different donors ± SE. ( E ) Role of GP and TLR4 signaling in induction of autophagy: Jukat cells were incubated with EBOV VLPs, VLPs lacking EBOV GP (VLP-ΔGP) or without VLPs with or without the TLR4 inhibitor CLI-095: representative primary LC3 flow cytometry data (left) and mean LC3 values based on triplicate samples ± SE. The gate indicates the position of LC3-positive cells based on the lack of staining with isotype control antibodies. ( F ) Imaging flow cytometry analysis of autophagy-specific granules of p62-RFP aggregates by an indicator Jurkat cell line. Top: representative images. Bottom: normalized levels of p62-RFP aggregates within the cell. A,B,C: representative of one of two independent experiments. D: representative of one of three independent experiments performed in triplicate samples; histogram is representative of individual samples from one experiment. E: representative images from one of two independent experiments. * P

    Journal: PLoS Pathogens

    Article Title: Ebola virus-mediated T-lymphocyte depletion is the result of an abortive infection

    doi: 10.1371/journal.ppat.1008068

    Figure Lengend Snippet: Abortive infection of T-cells results in ER-stress induced autophagy. ( A ) Flow cytometry analysis of intracellular Ca 2+ levels in primary CD4 + T-cells exposed to ionomycin, CD3/CD28 beads or EBOV VLPs for 10 min. Top dot plots results represent bound Ca 2+ (X axis) and free Ca 2+ (Y axis). Bottom dot plots represent the ratio of free Ca 2+ /Bound Ca 2+ over time during the 10 min stimulation. ( B ) Top: Western blot analysis of isolated CD4 + T-cells exposed to EBOV at various MOIs or mock-infected cells (M) for markers of ER-stress phospho-IRE1a and XBP1, as well as dephosphorylated form of IRE1a. Bottom: Western blot analysis of isolated CD4 + T-cells exposed to purified EBOV for XBP1. ( C ) Western blot analysis of phosphorylated eIF2a (P-eIF2a) in CD4 + T-cells exposed to EBOV in the presence or absence 1E7-03, okadaic acid (OA), a Lck inhibitor PP2 or a PI3K inhibitor Ly294. The ratio of phospho-eIF2a to tubulin is indicated below the Western blot images. ( D ) The induction of autophagy assessed by staining for LC3 in CD4 + T-cells exposed to EBOV or treated with rapamycin. Left: representative primary flow cytometry data. The gate indicates the position of LC3-positive cells based on the lack of staining with isotype control antibodies. Right: mean values based on triplicate samples from two different donors ± SE. ( E ) Role of GP and TLR4 signaling in induction of autophagy: Jukat cells were incubated with EBOV VLPs, VLPs lacking EBOV GP (VLP-ΔGP) or without VLPs with or without the TLR4 inhibitor CLI-095: representative primary LC3 flow cytometry data (left) and mean LC3 values based on triplicate samples ± SE. The gate indicates the position of LC3-positive cells based on the lack of staining with isotype control antibodies. ( F ) Imaging flow cytometry analysis of autophagy-specific granules of p62-RFP aggregates by an indicator Jurkat cell line. Top: representative images. Bottom: normalized levels of p62-RFP aggregates within the cell. A,B,C: representative of one of two independent experiments. D: representative of one of three independent experiments performed in triplicate samples; histogram is representative of individual samples from one experiment. E: representative images from one of two independent experiments. * P

    Article Snippet: Flow cytometry based analysis of autophagy was performed using the FlowCellect Autophagy LC3 Antibody-based Assay kit (Millipore) according to the manufacturer’s recommendations.

    Techniques: Infection, Flow Cytometry, Cytometry, Western Blot, Isolation, Purification, Staining, Incubation, Imaging

    CD4 + T-cells express a cellular restriction factor that blocks EBOV replication. ( A ) Flow cytometry analysis of fusion cells generated from DiL-labeled Jurkat cells and DiD-labeled Huh7 cells infected with EBOV-GFP. ( B ) Mean percentages of GFP + cells based on triplicate samples ±SE. ( C ) Mean relative binding of EBOV based on triplicate samples ±SE. ( D ) Ratio cRNA to gRNA based on triplicate samples ±SE. ( E ) Ratio mRNA to gRNA based on triplicate samples ±SE. B: representative of one of 3 independent experiments. C, D, E: representative of one of two independent experiments. * P

    Journal: PLoS Pathogens

    Article Title: Ebola virus-mediated T-lymphocyte depletion is the result of an abortive infection

    doi: 10.1371/journal.ppat.1008068

    Figure Lengend Snippet: CD4 + T-cells express a cellular restriction factor that blocks EBOV replication. ( A ) Flow cytometry analysis of fusion cells generated from DiL-labeled Jurkat cells and DiD-labeled Huh7 cells infected with EBOV-GFP. ( B ) Mean percentages of GFP + cells based on triplicate samples ±SE. ( C ) Mean relative binding of EBOV based on triplicate samples ±SE. ( D ) Ratio cRNA to gRNA based on triplicate samples ±SE. ( E ) Ratio mRNA to gRNA based on triplicate samples ±SE. B: representative of one of 3 independent experiments. C, D, E: representative of one of two independent experiments. * P

    Article Snippet: Flow cytometry based analysis of autophagy was performed using the FlowCellect Autophagy LC3 Antibody-based Assay kit (Millipore) according to the manufacturer’s recommendations.

    Techniques: Flow Cytometry, Cytometry, Generated, Labeling, Infection, Binding Assay

    EBOV viral proteins are readily detectable in CD4 + T-cells. ( A ) Confocal microscopy analysis of EBOV-exposed CD4 + T-cells for colocalization of the late-endosomal marker Rab7 (green), the lysosomal marker, LC3 (yellow) and EBOV antigens (red); nuclei are stained with DAPI (blue). ( B ) Dual immuno-gold labeling TEM of CD4 + T-cells exposed to EBOV: immunostained for CD3 with ~40 nm gold particles (black arrows) and for EBOV antigens with ~15 nm gold particles (red arrows). ( C ) Western blot analysis of EBOV VP40 and NP proteins in CD4 + T-cells incubated with EBOV for 2 or 5 days. (D) Western blot analysis of EBOV VP40 and NP proteins in EBOV-exposed Jurkat cells transfected with siRNAs targeting VP40 and NP. ( E ) Flow cytometry analysis of Jurkat cells and Huh7 cells incubated with EBOV. ( F,G ) Percentages of EBOV-positive Huh7 (F) and Jurkat (G) cells determined by flow cytometry, mean values based on triplicate samples ±SE. ** P

    Journal: PLoS Pathogens

    Article Title: Ebola virus-mediated T-lymphocyte depletion is the result of an abortive infection

    doi: 10.1371/journal.ppat.1008068

    Figure Lengend Snippet: EBOV viral proteins are readily detectable in CD4 + T-cells. ( A ) Confocal microscopy analysis of EBOV-exposed CD4 + T-cells for colocalization of the late-endosomal marker Rab7 (green), the lysosomal marker, LC3 (yellow) and EBOV antigens (red); nuclei are stained with DAPI (blue). ( B ) Dual immuno-gold labeling TEM of CD4 + T-cells exposed to EBOV: immunostained for CD3 with ~40 nm gold particles (black arrows) and for EBOV antigens with ~15 nm gold particles (red arrows). ( C ) Western blot analysis of EBOV VP40 and NP proteins in CD4 + T-cells incubated with EBOV for 2 or 5 days. (D) Western blot analysis of EBOV VP40 and NP proteins in EBOV-exposed Jurkat cells transfected with siRNAs targeting VP40 and NP. ( E ) Flow cytometry analysis of Jurkat cells and Huh7 cells incubated with EBOV. ( F,G ) Percentages of EBOV-positive Huh7 (F) and Jurkat (G) cells determined by flow cytometry, mean values based on triplicate samples ±SE. ** P

    Article Snippet: Flow cytometry based analysis of autophagy was performed using the FlowCellect Autophagy LC3 Antibody-based Assay kit (Millipore) according to the manufacturer’s recommendations.

    Techniques: Confocal Microscopy, Marker, Staining, Labeling, Transmission Electron Microscopy, Western Blot, Incubation, Transfection, Flow Cytometry, Cytometry

    The limited EBOV replication in CD4 + T-cells is not related to the anergic state. Flow cytometry analysis of CD4 + T cells from a representative donor exposed to EBOV at a MOI of 3 or 10 PFU/cell for 3 or 5 days. Cells were stained, and viable cell populations negative for LIVE/DEAD Fixable Aqua Dead Cell Stain were gated and analyzed for Ki-67 and for cell division. Left: representative primary flow cytometry data. Right: mean values based on triplicate samples ±SE, the MOI is indicated under the bars. ( A ) Analysis of for the activation marker Ki-67. ( B ) Analysis of proliferation by pre-staining of cells with the permanent dye CellTrace. Statistical differences compared to the day 0 values based on triplicate samples: *** P

    Journal: PLoS Pathogens

    Article Title: Ebola virus-mediated T-lymphocyte depletion is the result of an abortive infection

    doi: 10.1371/journal.ppat.1008068

    Figure Lengend Snippet: The limited EBOV replication in CD4 + T-cells is not related to the anergic state. Flow cytometry analysis of CD4 + T cells from a representative donor exposed to EBOV at a MOI of 3 or 10 PFU/cell for 3 or 5 days. Cells were stained, and viable cell populations negative for LIVE/DEAD Fixable Aqua Dead Cell Stain were gated and analyzed for Ki-67 and for cell division. Left: representative primary flow cytometry data. Right: mean values based on triplicate samples ±SE, the MOI is indicated under the bars. ( A ) Analysis of for the activation marker Ki-67. ( B ) Analysis of proliferation by pre-staining of cells with the permanent dye CellTrace. Statistical differences compared to the day 0 values based on triplicate samples: *** P

    Article Snippet: Flow cytometry based analysis of autophagy was performed using the FlowCellect Autophagy LC3 Antibody-based Assay kit (Millipore) according to the manufacturer’s recommendations.

    Techniques: Flow Cytometry, Cytometry, Staining, Activation Assay, Marker

    L . major infects newly recruited monocyte-derived cells independently of their differentiation stage. (A) Flow cytometry analysis of C57BL/6 (CD45.2) mice infected with Lm SWITCH , adoptively transferred 2 or 5 days before analysis with CD45.1 bone marrow cells. Gating strategy on CD45.1 + (newly recruited) and CD45.2 + (recipient) infected and non-infected cells. (B) Quantification of infected (red) and non-infected (blue) CD11c + F4/80 + double positive (upper panel) and CD11c + F4/80 - single positive (lower panel) among newly recruited cells at day 2 and day 5 post adoptive transfer. (C) Analysis as in (B) for recipient cells. (D) Experimental strategy to identify newly recruited cells present for 0–2 versus 3–5 days post adoptive transfer. Mice infected with non-fluorescent L . major wild type received mKikume-expressing bone marrow cells 5 days prior to analysis. Photoconversion at 3 days post transfer identifies cells recruited to the site of infection until day 3 in mKikume red fluorescence, while cells recruited after photoconversion (0–2 days prior to analysis) exhibit green mKikume fluorescence. (E) After gating on CD45 + cells, all adoptively transferred cells are identified by total mKikume fluorescence. Transferred cells which were at the infection site since 0–2 days (showing no red fluorescence) can be clearly distinguished from cells that were recruited to the site of infection 3–5 days before analysis (and thus photoconverted, showing a high red fluorescence). The two populations were analyzed for CD11c and F4/80 expression. (F) Quantitative analysis of CD11c + F4/80 + double positive (upper panel) and CD11c + F4/80 - single positive (lower panel) present at the site of infection for 0–2 days (green) and 3–5 days (red), respectively. Each dot represents one mouse ear. Horizontal bars denote the mean. *p

    Journal: PLoS Pathogens

    Article Title: CD11c-expressing Ly6C+CCR2+ monocytes constitute a reservoir for efficient Leishmania proliferation and cell-to-cell transmission

    doi: 10.1371/journal.ppat.1007374

    Figure Lengend Snippet: L . major infects newly recruited monocyte-derived cells independently of their differentiation stage. (A) Flow cytometry analysis of C57BL/6 (CD45.2) mice infected with Lm SWITCH , adoptively transferred 2 or 5 days before analysis with CD45.1 bone marrow cells. Gating strategy on CD45.1 + (newly recruited) and CD45.2 + (recipient) infected and non-infected cells. (B) Quantification of infected (red) and non-infected (blue) CD11c + F4/80 + double positive (upper panel) and CD11c + F4/80 - single positive (lower panel) among newly recruited cells at day 2 and day 5 post adoptive transfer. (C) Analysis as in (B) for recipient cells. (D) Experimental strategy to identify newly recruited cells present for 0–2 versus 3–5 days post adoptive transfer. Mice infected with non-fluorescent L . major wild type received mKikume-expressing bone marrow cells 5 days prior to analysis. Photoconversion at 3 days post transfer identifies cells recruited to the site of infection until day 3 in mKikume red fluorescence, while cells recruited after photoconversion (0–2 days prior to analysis) exhibit green mKikume fluorescence. (E) After gating on CD45 + cells, all adoptively transferred cells are identified by total mKikume fluorescence. Transferred cells which were at the infection site since 0–2 days (showing no red fluorescence) can be clearly distinguished from cells that were recruited to the site of infection 3–5 days before analysis (and thus photoconverted, showing a high red fluorescence). The two populations were analyzed for CD11c and F4/80 expression. (F) Quantitative analysis of CD11c + F4/80 + double positive (upper panel) and CD11c + F4/80 - single positive (lower panel) present at the site of infection for 0–2 days (green) and 3–5 days (red), respectively. Each dot represents one mouse ear. Horizontal bars denote the mean. *p

    Article Snippet: Flow cytometry Ears of mice were separated in two sheets (ventral and dorsal) using forceps and enzymatically digested in RPMI 1640 medium containing 1 mg/ml collagenase (Sigma) and 50 ng/ml DNase (Sigma-Aldrich) for 60 min at 600 rpm and 37°C, and passed through a 70 μm cell strainer.

    Techniques: Derivative Assay, Flow Cytometry, Cytometry, Mouse Assay, Infection, Adoptive Transfer Assay, Expressing, Fluorescence

    CD11c-expressing Ly6C + CCR2 + monocytes are the main cellular niche of high L . major proliferation. (A-D) C57BL/6 mice were infected for 3 weeks with monofluorescent DsRed-expressing L . major compatible with multicolor flow cytometry. (A) Among CD45 + cells, mainly the CD11b + cells were found to be infected with L . major . (B-C) Cell populations defined by CD11c and F4/80 expression were further analyzed with respect to expression of CCR2, Ly6C, and MHC class II. Red plots and histograms, infected cells; black plots and histograms, all cells. (D) Quantitative analysis of marker expression shown in (C) for infected cells. Vertical bars represent the mean. *p

    Journal: PLoS Pathogens

    Article Title: CD11c-expressing Ly6C+CCR2+ monocytes constitute a reservoir for efficient Leishmania proliferation and cell-to-cell transmission

    doi: 10.1371/journal.ppat.1007374

    Figure Lengend Snippet: CD11c-expressing Ly6C + CCR2 + monocytes are the main cellular niche of high L . major proliferation. (A-D) C57BL/6 mice were infected for 3 weeks with monofluorescent DsRed-expressing L . major compatible with multicolor flow cytometry. (A) Among CD45 + cells, mainly the CD11b + cells were found to be infected with L . major . (B-C) Cell populations defined by CD11c and F4/80 expression were further analyzed with respect to expression of CCR2, Ly6C, and MHC class II. Red plots and histograms, infected cells; black plots and histograms, all cells. (D) Quantitative analysis of marker expression shown in (C) for infected cells. Vertical bars represent the mean. *p

    Article Snippet: Flow cytometry Ears of mice were separated in two sheets (ventral and dorsal) using forceps and enzymatically digested in RPMI 1640 medium containing 1 mg/ml collagenase (Sigma) and 50 ng/ml DNase (Sigma-Aldrich) for 60 min at 600 rpm and 37°C, and passed through a 70 μm cell strainer.

    Techniques: Expressing, Mouse Assay, Infection, Flow Cytometry, Cytometry, Marker

    CD11c + cells represent a niche for maintaining a high L . major burden. (A-B) C57BL/6 mice ears were infected intradermally with Lm SWITCH . 3 weeks post infection, parasites in the mouse ear were photoconverted and 48h later ears were harvested for flow cytometry analysis of intracellular L . major proliferation. (A) Strategy to identify L . major -infected cells (large plot) and CD11c + and F4/80 + cell subsets (B) using controls infected with non-fluorescent L . major wildtype parasites (A, middle row, small plot) and isotype controls (A, lower row, small plots). (C) Comparison of proliferation rates of Lm SWITCH located in the different cell compartments defined by F4/80 and CD11c. Vertical bars denote the mean, and shaded red boxes the standard deviation. (D) Quantitative analysis of the proliferation rates of Lm SWITCH within the different cell populations shown in B-C. Each dot represents one mouse ear. Relative proliferation indices were obtained by normalization of the subpopulation’s proliferation index to the overall mean proliferation index within each sample. Vertical bars represent the mean. *p

    Journal: PLoS Pathogens

    Article Title: CD11c-expressing Ly6C+CCR2+ monocytes constitute a reservoir for efficient Leishmania proliferation and cell-to-cell transmission

    doi: 10.1371/journal.ppat.1007374

    Figure Lengend Snippet: CD11c + cells represent a niche for maintaining a high L . major burden. (A-B) C57BL/6 mice ears were infected intradermally with Lm SWITCH . 3 weeks post infection, parasites in the mouse ear were photoconverted and 48h later ears were harvested for flow cytometry analysis of intracellular L . major proliferation. (A) Strategy to identify L . major -infected cells (large plot) and CD11c + and F4/80 + cell subsets (B) using controls infected with non-fluorescent L . major wildtype parasites (A, middle row, small plot) and isotype controls (A, lower row, small plots). (C) Comparison of proliferation rates of Lm SWITCH located in the different cell compartments defined by F4/80 and CD11c. Vertical bars denote the mean, and shaded red boxes the standard deviation. (D) Quantitative analysis of the proliferation rates of Lm SWITCH within the different cell populations shown in B-C. Each dot represents one mouse ear. Relative proliferation indices were obtained by normalization of the subpopulation’s proliferation index to the overall mean proliferation index within each sample. Vertical bars represent the mean. *p

    Article Snippet: Flow cytometry Ears of mice were separated in two sheets (ventral and dorsal) using forceps and enzymatically digested in RPMI 1640 medium containing 1 mg/ml collagenase (Sigma) and 50 ng/ml DNase (Sigma-Aldrich) for 60 min at 600 rpm and 37°C, and passed through a 70 μm cell strainer.

    Techniques: Mouse Assay, Infection, Flow Cytometry, Cytometry, Standard Deviation

    Knockdown of Beclin1 inhibits tumor growth and alters TAMs polarization. a BECN1-KD B16F10 and Ctrl-B16F10 cells were s.c. implanted to C57BL/6 mice ( n = 6 per group). Tumor area was measured at the described days. b Tumor bearing mice were euthanized around day 15, total TAMs (F4/80 + CD11b + ) were gated and analyzed for mean fluorescent intensity (MFI) of CD206, PD-L1, CD86 and MHC II expression by flow cytometry. Events are gated on singlet, live and CD45 + tumor infiltrating cells. c TILs were stimulated with Cell Stimulation Cocktail for 5 h, the frequency of CD4 + IFN-γ + and CD8 + IFN-γ + T cells was determined by intracellular staining. Single suspension cells from dLNs and spleens were stimulated with mitomycin C inactivated B16F10 cells at a ratio of 30:1 for 21 h, breferdin A plus monensin was added for the last 5 h of culture, and IFN-γ production was determined by flow cytometry. d T cells from TILs, dLNs and spleens were tested for Ki-67 expression by intracellular staining. e B16F10 cells were mixed with WT or PD-L1 −/− BMDMs (2:1) treated with or without TRAPs and then injected s.c. to C57BL/6 mice (n = 6 per group). Tumor growth was monitored at the indicated days. Data (mean ± SEM) are representative of three independent experiments. * p

    Journal: Journal for Immunotherapy of Cancer

    Article Title: Tumor cell-released autophagosomes (TRAPs) promote immunosuppression through induction of M2-like macrophages with increased expression of PD-L1

    doi: 10.1186/s40425-018-0452-5

    Figure Lengend Snippet: Knockdown of Beclin1 inhibits tumor growth and alters TAMs polarization. a BECN1-KD B16F10 and Ctrl-B16F10 cells were s.c. implanted to C57BL/6 mice ( n = 6 per group). Tumor area was measured at the described days. b Tumor bearing mice were euthanized around day 15, total TAMs (F4/80 + CD11b + ) were gated and analyzed for mean fluorescent intensity (MFI) of CD206, PD-L1, CD86 and MHC II expression by flow cytometry. Events are gated on singlet, live and CD45 + tumor infiltrating cells. c TILs were stimulated with Cell Stimulation Cocktail for 5 h, the frequency of CD4 + IFN-γ + and CD8 + IFN-γ + T cells was determined by intracellular staining. Single suspension cells from dLNs and spleens were stimulated with mitomycin C inactivated B16F10 cells at a ratio of 30:1 for 21 h, breferdin A plus monensin was added for the last 5 h of culture, and IFN-γ production was determined by flow cytometry. d T cells from TILs, dLNs and spleens were tested for Ki-67 expression by intracellular staining. e B16F10 cells were mixed with WT or PD-L1 −/− BMDMs (2:1) treated with or without TRAPs and then injected s.c. to C57BL/6 mice (n = 6 per group). Tumor growth was monitored at the indicated days. Data (mean ± SEM) are representative of three independent experiments. * p

    Article Snippet: For flow cytometry identification, TRAPs were stained with rabbit anti-LC3B antibody (Sigma), followed by R-PE conjugated anti-Rabbit IgG secondary antibody (proteintech), or stained with PE-LC3B mAb (Cell Signaling Technology).

    Techniques: Mouse Assay, Expressing, Flow Cytometry, Cytometry, Cell Stimulation, Staining, Injection

    TRAPs derived from malignant pleural effusions or ascites of cancer patients regulate monocytes polarization. a , b The MFIs of ( a) CD163 and ( b ) PD-L1 on CD14 + monocytes from peripheral blood of cancer patients (CPs) ( n = 14) and healthy donors (HDs) ( n = 8) were assessed by flow cytometry. c PBMCs from CPs and HDs were stimulated with LPS (100 ng/ml) for 18 h, breferdin A plus monensin was added for the last 6 h of culture, and the frequency of CD14 + IL-10 + was determined by flow cytometry. d , e The graphs showed the correlation between concentration of TRAP and MFI of PD-L1 on CD14 + monocytes and total IL-10 in malignant pleural effusions or ascites of cancer patients ( n = 20). f-h Purified CD14 + monocytes from healthy donors were treated with TRAPs (5 μg/ml) derived from pleural effusions or ascites of cancer patients ( n = 10) for 3 d. f , g The expression of CD163, PD-L1, CD86 and HLA-DR was detected by flow cytometry. h The amount of IL-10 in the supernatant was assessed by ELISA. i Analysis of T cell proliferation by flow cytometry. Monocytes were left untreated or pretreated with TRAPs for 3 d, then incubated with CFSE-labeled autologous T cells (1:3) for 5 d in the presence of anti-CD3 and anti-CD28, or monocytes and T cells were separated by a transwell chamber. j , k Monocytes were left untreated or pretreated with TRAPs from MDA-MB-231 cells (c-TRAP) and cancer patient (p-TRAP) for 3 d, then incubated with autologous T cells (1:3) for 20 h in the presence of immobilized anti-CD3. j CD25 expression on T cells was examined by flow cytometry. k Breferdin A plus monensin was added during the last 8 h of culture. The percentage of CD4 + IFN-γ + and CD8 + IFN-γ + T cells and total IFN-γ secretion in the supernatant was detected by flow cytometry and ELISA, respectively. Data (mean ± SEM) are representative of three independent experiments. * p

    Journal: Journal for Immunotherapy of Cancer

    Article Title: Tumor cell-released autophagosomes (TRAPs) promote immunosuppression through induction of M2-like macrophages with increased expression of PD-L1

    doi: 10.1186/s40425-018-0452-5

    Figure Lengend Snippet: TRAPs derived from malignant pleural effusions or ascites of cancer patients regulate monocytes polarization. a , b The MFIs of ( a) CD163 and ( b ) PD-L1 on CD14 + monocytes from peripheral blood of cancer patients (CPs) ( n = 14) and healthy donors (HDs) ( n = 8) were assessed by flow cytometry. c PBMCs from CPs and HDs were stimulated with LPS (100 ng/ml) for 18 h, breferdin A plus monensin was added for the last 6 h of culture, and the frequency of CD14 + IL-10 + was determined by flow cytometry. d , e The graphs showed the correlation between concentration of TRAP and MFI of PD-L1 on CD14 + monocytes and total IL-10 in malignant pleural effusions or ascites of cancer patients ( n = 20). f-h Purified CD14 + monocytes from healthy donors were treated with TRAPs (5 μg/ml) derived from pleural effusions or ascites of cancer patients ( n = 10) for 3 d. f , g The expression of CD163, PD-L1, CD86 and HLA-DR was detected by flow cytometry. h The amount of IL-10 in the supernatant was assessed by ELISA. i Analysis of T cell proliferation by flow cytometry. Monocytes were left untreated or pretreated with TRAPs for 3 d, then incubated with CFSE-labeled autologous T cells (1:3) for 5 d in the presence of anti-CD3 and anti-CD28, or monocytes and T cells were separated by a transwell chamber. j , k Monocytes were left untreated or pretreated with TRAPs from MDA-MB-231 cells (c-TRAP) and cancer patient (p-TRAP) for 3 d, then incubated with autologous T cells (1:3) for 20 h in the presence of immobilized anti-CD3. j CD25 expression on T cells was examined by flow cytometry. k Breferdin A plus monensin was added during the last 8 h of culture. The percentage of CD4 + IFN-γ + and CD8 + IFN-γ + T cells and total IFN-γ secretion in the supernatant was detected by flow cytometry and ELISA, respectively. Data (mean ± SEM) are representative of three independent experiments. * p

    Article Snippet: For flow cytometry identification, TRAPs were stained with rabbit anti-LC3B antibody (Sigma), followed by R-PE conjugated anti-Rabbit IgG secondary antibody (proteintech), or stained with PE-LC3B mAb (Cell Signaling Technology).

    Techniques: Derivative Assay, Flow Cytometry, Cytometry, Concentration Assay, Purification, Expressing, Enzyme-linked Immunosorbent Assay, Incubation, Labeling, Multiple Displacement Amplification

    TRAPs polarize macrophages toward an M2-like phenotype in vitro and in vivo. a Confocal images of BMDMs treated with CFSE-labeled TRAPs (green). After incubation with TRAPs (10 μg/ml) for 0.5 h, BMDMs were stained with PE-F4/80 antibody (red) and DAPI (blue). Scale bar, 10 μm. b Expression analysis of CD206, PD-L1, CD86 and MHC II by flow cytometry. BMDMs were stimulated with LPS (100 ng/ml) + IFN-γ (20 ng/ml), IL-4 (20 ng/ml) or TRAPs (10 μg/ml) for 48, 48 or 72 h, respectively. c Flow cytometry analysis of PD-L2, B7-H2, B7-H3, B7-H4, Tim-4 and VISTA for BMDMs after incubating with TRAPs for 48 h. d Expression analysis of NOS2 and Arg1 mRNA in BMDMs treated with TRAPs (10 μg/ml) for 6 h by qRT-PCR. e ELISA detection of IL-1β, IL-6, IL-10 and IL-12p70 produced by BMDMs exposed to TRAPs (10 μg/ml) for 72 h. f-h Mice ( n = 3) were intraperitoneally injected with four different doses of TRAPs (0, 10, 30 and 100 μg) at day 0 and the phenotype of peritoneal macrophages was analyzed at day 3. Expression of CD206 ( f ) and PD-L1 ( g ) on macrophages (F4/80 + CD11b + ) was determined by flow cytometry. h mRNA expression level of IL-10 and Arg1 in purified macrophages was detected by qRT-PCR. Results are representative of three independent experiments. * p

    Journal: Journal for Immunotherapy of Cancer

    Article Title: Tumor cell-released autophagosomes (TRAPs) promote immunosuppression through induction of M2-like macrophages with increased expression of PD-L1

    doi: 10.1186/s40425-018-0452-5

    Figure Lengend Snippet: TRAPs polarize macrophages toward an M2-like phenotype in vitro and in vivo. a Confocal images of BMDMs treated with CFSE-labeled TRAPs (green). After incubation with TRAPs (10 μg/ml) for 0.5 h, BMDMs were stained with PE-F4/80 antibody (red) and DAPI (blue). Scale bar, 10 μm. b Expression analysis of CD206, PD-L1, CD86 and MHC II by flow cytometry. BMDMs were stimulated with LPS (100 ng/ml) + IFN-γ (20 ng/ml), IL-4 (20 ng/ml) or TRAPs (10 μg/ml) for 48, 48 or 72 h, respectively. c Flow cytometry analysis of PD-L2, B7-H2, B7-H3, B7-H4, Tim-4 and VISTA for BMDMs after incubating with TRAPs for 48 h. d Expression analysis of NOS2 and Arg1 mRNA in BMDMs treated with TRAPs (10 μg/ml) for 6 h by qRT-PCR. e ELISA detection of IL-1β, IL-6, IL-10 and IL-12p70 produced by BMDMs exposed to TRAPs (10 μg/ml) for 72 h. f-h Mice ( n = 3) were intraperitoneally injected with four different doses of TRAPs (0, 10, 30 and 100 μg) at day 0 and the phenotype of peritoneal macrophages was analyzed at day 3. Expression of CD206 ( f ) and PD-L1 ( g ) on macrophages (F4/80 + CD11b + ) was determined by flow cytometry. h mRNA expression level of IL-10 and Arg1 in purified macrophages was detected by qRT-PCR. Results are representative of three independent experiments. * p

    Article Snippet: For flow cytometry identification, TRAPs were stained with rabbit anti-LC3B antibody (Sigma), followed by R-PE conjugated anti-Rabbit IgG secondary antibody (proteintech), or stained with PE-LC3B mAb (Cell Signaling Technology).

    Techniques: In Vitro, In Vivo, Labeling, Incubation, Staining, Expressing, Flow Cytometry, Cytometry, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Produced, Mouse Assay, Injection, Purification

    TRAPs exposed macrophages inhibit T cell proliferation via PD-L1 and IL-10. a-d Representative determination of T cell proliferation by flow cytometry. a , b , d CFSE-labeled purified CD3 + T cells were activated by immobilized anti-CD3 plus soluble anti-CD28 mAb, and were either cultured alone or cocultured with BMDMs pretreated with or without TRAPs at a ratio of 5:1 for 3 d. b A transwell chamber was used to separate BMDMs from T cells. d BMDMs were cocultured with T cells in the presence of anti-PD-L1 mAb, anti-IL-10 mAb or control IgG. c BMDMs were left untreated or pretreated with 10 μg/ml B16F10 TRAPs or B16F10-OVA TRAPs for 2 d, and loaded with OVA 257–264 peptide SIINFEKL (1 μg/ml) for 2 h, washed, were then incubated with CFSE-labeled OT-I splenocytes for 3 d at a ratio of 1:20. Data are representative of three independent experiments. * p

    Journal: Journal for Immunotherapy of Cancer

    Article Title: Tumor cell-released autophagosomes (TRAPs) promote immunosuppression through induction of M2-like macrophages with increased expression of PD-L1

    doi: 10.1186/s40425-018-0452-5

    Figure Lengend Snippet: TRAPs exposed macrophages inhibit T cell proliferation via PD-L1 and IL-10. a-d Representative determination of T cell proliferation by flow cytometry. a , b , d CFSE-labeled purified CD3 + T cells were activated by immobilized anti-CD3 plus soluble anti-CD28 mAb, and were either cultured alone or cocultured with BMDMs pretreated with or without TRAPs at a ratio of 5:1 for 3 d. b A transwell chamber was used to separate BMDMs from T cells. d BMDMs were cocultured with T cells in the presence of anti-PD-L1 mAb, anti-IL-10 mAb or control IgG. c BMDMs were left untreated or pretreated with 10 μg/ml B16F10 TRAPs or B16F10-OVA TRAPs for 2 d, and loaded with OVA 257–264 peptide SIINFEKL (1 μg/ml) for 2 h, washed, were then incubated with CFSE-labeled OT-I splenocytes for 3 d at a ratio of 1:20. Data are representative of three independent experiments. * p

    Article Snippet: For flow cytometry identification, TRAPs were stained with rabbit anti-LC3B antibody (Sigma), followed by R-PE conjugated anti-Rabbit IgG secondary antibody (proteintech), or stained with PE-LC3B mAb (Cell Signaling Technology).

    Techniques: Flow Cytometry, Cytometry, Labeling, Purification, Cell Culture, Incubation

    p38-STAT3 signaling in BMDMs and protein fraction in TRAPs are essential for induction of PD-L1 and IL-10. a BMDMs were exposed to TRAPs (10 μg/ml) at indicated time points. Cell lysates were analyzed for p38, p-p38, STAT3 and p-STAT3 by western blot. GAPDH was used as a loading control. b BMDMs were pretreated with p38 inhibitor SB203580 (3 μM) for 1 h, and then co-incubated with TRAPs (10 μg/ml) for 4 h. Expression of STAT3 and p-STAT3 was detected by western blot. c BMDMs were exposed to SB203580 at described concentrations for 1 h, and followed by incubation with TRAPs (10 μg/ml) for 72 h. PD-L1 expression was determined by flow cytometry, and ( d ) the production of IL-10 was assessed by ELISA. e BMDMs were pretreated with STAT3 inhibitor Stattic (1 and 3 μM) for 1 h, and then stimulated with TRAPs (10 μg/ml) for 72 h. PD-L1 expression and ( f ) IL-10 secretion was determined by flow cytometry and ELISA, respectively. g TRAPs were pretreated with Proteinase K for 2 h at 55 °C, DNase I for 1 h at 37 °C or RNase for 3 h at 37 °C, respectively, followed by incubation with BMDMs for 72 h. PD-L1 was evaluated by flow cytometry, and ( h ) IL-10 was tested by ELISA. Data are shown as mean ± SEM, and are representative of three independent experiments. *** p

    Journal: Journal for Immunotherapy of Cancer

    Article Title: Tumor cell-released autophagosomes (TRAPs) promote immunosuppression through induction of M2-like macrophages with increased expression of PD-L1

    doi: 10.1186/s40425-018-0452-5

    Figure Lengend Snippet: p38-STAT3 signaling in BMDMs and protein fraction in TRAPs are essential for induction of PD-L1 and IL-10. a BMDMs were exposed to TRAPs (10 μg/ml) at indicated time points. Cell lysates were analyzed for p38, p-p38, STAT3 and p-STAT3 by western blot. GAPDH was used as a loading control. b BMDMs were pretreated with p38 inhibitor SB203580 (3 μM) for 1 h, and then co-incubated with TRAPs (10 μg/ml) for 4 h. Expression of STAT3 and p-STAT3 was detected by western blot. c BMDMs were exposed to SB203580 at described concentrations for 1 h, and followed by incubation with TRAPs (10 μg/ml) for 72 h. PD-L1 expression was determined by flow cytometry, and ( d ) the production of IL-10 was assessed by ELISA. e BMDMs were pretreated with STAT3 inhibitor Stattic (1 and 3 μM) for 1 h, and then stimulated with TRAPs (10 μg/ml) for 72 h. PD-L1 expression and ( f ) IL-10 secretion was determined by flow cytometry and ELISA, respectively. g TRAPs were pretreated with Proteinase K for 2 h at 55 °C, DNase I for 1 h at 37 °C or RNase for 3 h at 37 °C, respectively, followed by incubation with BMDMs for 72 h. PD-L1 was evaluated by flow cytometry, and ( h ) IL-10 was tested by ELISA. Data are shown as mean ± SEM, and are representative of three independent experiments. *** p

    Article Snippet: For flow cytometry identification, TRAPs were stained with rabbit anti-LC3B antibody (Sigma), followed by R-PE conjugated anti-Rabbit IgG secondary antibody (proteintech), or stained with PE-LC3B mAb (Cell Signaling Technology).

    Techniques: Western Blot, Incubation, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    Immunosuppression mediated by TRAPs treated BMDMs is dependent on TLR4-MyD88 signaling. a Expression analysis of PD-L1 by flow cytometry. BMDMs derived from WT, TLR2 −/− , TLR4 −/− , or MyD88 −/− mice were incubated with TRAPs (10 μg/ml) for 48 h. b ELISA detection of IL-10 secreted from BMDMs exposed to TRAPs (10 μg/ml) for 72 h. c , d Detection of OT-I CD8 + T cell division by flow cytometry. BMDMs derived from WT, TLR2 −/− , TLR4 −/− , or MyD88 −/− mice were left untreated or pretreated with B16F10 TRAPs (10 μg/ml) for 2 d, and pulsed with peptide SIINFEKL (1 μg/ml) for 2 h, washed, and followed by coculture with CFSE-labeled OT-I splenocytes for 3 d at a ratio of 1:20. Data (mean ± SEM) are representative of three independent experiments. ** p

    Journal: Journal for Immunotherapy of Cancer

    Article Title: Tumor cell-released autophagosomes (TRAPs) promote immunosuppression through induction of M2-like macrophages with increased expression of PD-L1

    doi: 10.1186/s40425-018-0452-5

    Figure Lengend Snippet: Immunosuppression mediated by TRAPs treated BMDMs is dependent on TLR4-MyD88 signaling. a Expression analysis of PD-L1 by flow cytometry. BMDMs derived from WT, TLR2 −/− , TLR4 −/− , or MyD88 −/− mice were incubated with TRAPs (10 μg/ml) for 48 h. b ELISA detection of IL-10 secreted from BMDMs exposed to TRAPs (10 μg/ml) for 72 h. c , d Detection of OT-I CD8 + T cell division by flow cytometry. BMDMs derived from WT, TLR2 −/− , TLR4 −/− , or MyD88 −/− mice were left untreated or pretreated with B16F10 TRAPs (10 μg/ml) for 2 d, and pulsed with peptide SIINFEKL (1 μg/ml) for 2 h, washed, and followed by coculture with CFSE-labeled OT-I splenocytes for 3 d at a ratio of 1:20. Data (mean ± SEM) are representative of three independent experiments. ** p

    Article Snippet: For flow cytometry identification, TRAPs were stained with rabbit anti-LC3B antibody (Sigma), followed by R-PE conjugated anti-Rabbit IgG secondary antibody (proteintech), or stained with PE-LC3B mAb (Cell Signaling Technology).

    Techniques: Expressing, Flow Cytometry, Cytometry, Derivative Assay, Mouse Assay, Incubation, Enzyme-linked Immunosorbent Assay, Labeling

    DNA response induced by 5-Fu, Gyp and 5-Fu + Gyp. (A) DNA fragmentation was detected using flow cytometry after 5-Fu, Gyp and 5-Fu + Gyp treatment in SW-480 cells after 24 and 48 h. Histograms showed number of cell channels (vertical axis) vs. PI fluorescence (horizontal axis). (B) Comet assay was performed for detection of DNA damage after 5-Fu co-treated with Gyp. (C) The expression level of Ser139-Histone H2A.X was determined using western blotting. Experiments were done independently in triplicate per experimental point, and representative results were shown. All data are expressed as means ± SD of triplicates and p *

    Journal: PLoS ONE

    Article Title: Gypenosides Synergistically Enhances the Anti-Tumor Effect of 5-Fluorouracil on Colorectal Cancer In Vitro and In Vivo: A Role for Oxidative Stress-Mediated DNA Damage and p53 Activation

    doi: 10.1371/journal.pone.0137888

    Figure Lengend Snippet: DNA response induced by 5-Fu, Gyp and 5-Fu + Gyp. (A) DNA fragmentation was detected using flow cytometry after 5-Fu, Gyp and 5-Fu + Gyp treatment in SW-480 cells after 24 and 48 h. Histograms showed number of cell channels (vertical axis) vs. PI fluorescence (horizontal axis). (B) Comet assay was performed for detection of DNA damage after 5-Fu co-treated with Gyp. (C) The expression level of Ser139-Histone H2A.X was determined using western blotting. Experiments were done independently in triplicate per experimental point, and representative results were shown. All data are expressed as means ± SD of triplicates and p *

    Article Snippet: Cell cycle analysis SW-480 cells were seeded in 24-well plates and exposure to 5-Fu, Gyp or 5-Fu + Gyp for 24 and 48 h. Then the treated cells were harvested and disposed as following steps: washed twice with cold PBS, fixed with 70% ice-cold ethanol at -20°C overnight, washed twice with cold PBS, incubated with 100 mg/ml RNase A for 30 min at 37°C, after that stained with 50 mg/ml PI in the dark for 30 min and subjected to flow cytometry analysis.

    Techniques: Flow Cytometry, Cytometry, Fluorescence, Single Cell Gel Electrophoresis, Expressing, Western Blot

    The

    Journal: BMC Cell Biology

    Article Title: Multistable and multistep dynamics in neutrophil differentiation

    doi: 10.1186/1471-2121-7-11

    Figure Lengend Snippet: The "primed" and native populations are indistinguishable by CD11b or phosphorylated-Erk expression levels . A . The "primed" (green) and "native" (red) populations were measured for CD11b expression immediately after FACS sorting and found to be indistinguishable. For comparison, CD11b signal from the treated population before sorting is presented (black) showing a combination of CD11b Low and CD11b High cells. B . Similarly, intracellular phosphorylated-Erk (Erk-P) expression for "primed" (green) and "native" (red) populations as monitored by flow cytometry gave nearly identical results. In comparison, a sample treated with 1.3% DMSO for 7 days (black) revealed much larger variations in Erk-P expression.

    Article Snippet: Flow cytometry and Fluorescence Activated Cell Sorting (FACS) Flow cytometry was performed on a Guava-PCA microfluidic-based flow cytometer (GuavaTechnologies, Inc).

    Techniques: Expressing, FACS, Flow Cytometry, Cytometry

    The experiments outlined in Fig. 6 revealed that CD11b expression in DMSO-treated HL60 cells is a multi-step process involving an intermediate

    Journal: BMC Cell Biology

    Article Title: Multistable and multistep dynamics in neutrophil differentiation

    doi: 10.1186/1471-2121-7-11

    Figure Lengend Snippet: The experiments outlined in Fig. 6 revealed that CD11b expression in DMSO-treated HL60 cells is a multi-step process involving an intermediate "primed" state . ( A , B ). The "restimulated" (solid diamonds) and "twice restimulated" (solid circles) cells both showed a similar increased rate of CD11b expression (fraction of CD11b High cells) relative to the "native" (open triangles) or "untreated" (open squares) groups. ( C , D ). This accelerated kinetics was sustained for up to four days in DMSO-free medium but was gradually lost beyond four days. Duplicates are presented, where solid circles, open diamonds, open triangles, solid squares, and solid triangles (--) represent cells cultured for 1, 2(for C )/4(for D ), 5, 6, and 7 days in control medium before restimulation with 0.8% DMSO, respectively. Open squares indicate cells of the native population that have never been previously treated with DMSO nor FACS sorted. Each data point represents a flow cytometry measurement obtained from 10,000 cells.

    Article Snippet: Flow cytometry and Fluorescence Activated Cell Sorting (FACS) Flow cytometry was performed on a Guava-PCA microfluidic-based flow cytometer (GuavaTechnologies, Inc).

    Techniques: Expressing, Cell Culture, FACS, Flow Cytometry, Cytometry

    Experimental design for analyzing multi-dimensional dynamics of differentiation . Shaded block arrows represent cells exposed to 0.8% DMSO for the indicated durations while open arrows indicate

    Journal: BMC Cell Biology

    Article Title: Multistable and multistep dynamics in neutrophil differentiation

    doi: 10.1186/1471-2121-7-11

    Figure Lengend Snippet: Experimental design for analyzing multi-dimensional dynamics of differentiation . Shaded block arrows represent cells exposed to 0.8% DMSO for the indicated durations while open arrows indicate "untreated" cell cultures. Brackets indicate subpopulations sorted with FACS. The fraction of CD11b High cells was monitored by flow cytometry daily for 7 days for the "restimulated", "native" (mock-sorted and restimulated) and "untreated" groups. A CD11b Low fraction that was re-established upon restimulation with 0.8% DMSO for 4 days was also sorted, restimulated a second time ("twice restimulated"), and monitored by flow cytometry for 6 days.

    Article Snippet: Flow cytometry and Fluorescence Activated Cell Sorting (FACS) Flow cytometry was performed on a Guava-PCA microfluidic-based flow cytometer (GuavaTechnologies, Inc).

    Techniques: Blocking Assay, FACS, Flow Cytometry, Cytometry

    The expression of FN1BP1 induced G1 phase arrest. Synchronized by nocodazole for 20 h, Dox was added to half of the plates to induce the expression of FN1BP1 for 24 h. Following UV irradiation at 200 (×100 µJ/cm2) for 1 min, the cells were obtained after another 12-h incubation. Then the cells were collected and stained by propidium iodide (PI) for flow cytometry (FCM). (A–B) showed the representative figure of the result of FCM for DOX (−) and DOX (+), respectively. The statistical result is shown in (C). The statistical data demonstrate that more cells induced by Dox were arrested in the G1 phase compared with the non-Dox–induced cells. *P

    Journal: PLoS ONE

    Article Title: Identification of FN1BP1 as a Novel Cell Cycle Regulator through Modulating G1 Checkpoint in Human Hepatocarcinoma Hep3B Cells

    doi: 10.1371/journal.pone.0057574

    Figure Lengend Snippet: The expression of FN1BP1 induced G1 phase arrest. Synchronized by nocodazole for 20 h, Dox was added to half of the plates to induce the expression of FN1BP1 for 24 h. Following UV irradiation at 200 (×100 µJ/cm2) for 1 min, the cells were obtained after another 12-h incubation. Then the cells were collected and stained by propidium iodide (PI) for flow cytometry (FCM). (A–B) showed the representative figure of the result of FCM for DOX (−) and DOX (+), respectively. The statistical result is shown in (C). The statistical data demonstrate that more cells induced by Dox were arrested in the G1 phase compared with the non-Dox–induced cells. *P

    Article Snippet: For flow cytometry (FCM) analysis following the method described in previous studies , , the ethanol-fixed cells were digested by 1% RNase A (ribonuclease A, Sigma) for 30 min, stained with propidium iodide (PI) at 4°C, protected from light, filtered twice, then measured with a flow cytometer (FACS Calibur, Becton Dickinson, Franklin Lakes, NJ, USA).

    Techniques: Expressing, Irradiation, Incubation, Staining, Flow Cytometry, Cytometry

    Additional protein expression data. (A) Isotype controls used for flow cytometry plot gating in Figure 3B . (B) Expression of intestinal markers E-Cadherin, CDX2, and Villin is shown in primary hInEpC Donors B (top row) and C (bottom row).

    Journal: Frontiers in Pharmacology

    Article Title: Alternative functional in vitro models of human intestinal epithelia

    doi: 10.3389/fphar.2013.00079

    Figure Lengend Snippet: Additional protein expression data. (A) Isotype controls used for flow cytometry plot gating in Figure 3B . (B) Expression of intestinal markers E-Cadherin, CDX2, and Villin is shown in primary hInEpC Donors B (top row) and C (bottom row).

    Article Snippet: Flow cytometry Undifferentiated or Stage 1 iPSCs were detached by treatment with Accutase (Sigma-Aldrich; St. Louis, MO) and stained for viability by Near Infrared Live/Dead kit (Invitrogen; Carlsbad, CA) before fixation in Cytofix Buffer (BD Biosciences; San Jose, CA).

    Techniques: Expressing, Flow Cytometry, Cytometry

    Intestinal epithelial cell expression of FcRn. (A) Immunohistochemical analysis demonstrates iPSC-derived Stage 3 intestinal organoid expression and punctate localization of neonatal Fc Receptor expression in iPSC-derived organoids. Scale bar, 100 μm. (B) Flow cytometry analysis shows surface FcRn expression on Caco-2 (left) and primary hInEpCs (right, Donor A shown).

    Journal: Frontiers in Pharmacology

    Article Title: Alternative functional in vitro models of human intestinal epithelia

    doi: 10.3389/fphar.2013.00079

    Figure Lengend Snippet: Intestinal epithelial cell expression of FcRn. (A) Immunohistochemical analysis demonstrates iPSC-derived Stage 3 intestinal organoid expression and punctate localization of neonatal Fc Receptor expression in iPSC-derived organoids. Scale bar, 100 μm. (B) Flow cytometry analysis shows surface FcRn expression on Caco-2 (left) and primary hInEpCs (right, Donor A shown).

    Article Snippet: Flow cytometry Undifferentiated or Stage 1 iPSCs were detached by treatment with Accutase (Sigma-Aldrich; St. Louis, MO) and stained for viability by Near Infrared Live/Dead kit (Invitrogen; Carlsbad, CA) before fixation in Cytofix Buffer (BD Biosciences; San Jose, CA).

    Techniques: Expressing, Immunohistochemistry, Derivative Assay, Flow Cytometry, Cytometry

    T cell cytokine synthesis and proliferation in PTPN4/PTPN3 double-deficient mice. A) LN T cells were stimulated with the indicated concentrations of CD3 antibody and 0.75 µg/mL CD28 antibody. Concentrations of cytokines in supernatants were determined as in Figure 2 . Each bar represents the mean plus one standard deviation of triplicate determinations from one mouse. Differences between mice are not statistically significant (Student's T-test). Data are representative of six repeat experiments. B) Splenic T cells were labeled with CFSE and stimulated with 0.1 µg/mL CD3 antibody and 0.75 µg/mL CD28 antibody. After 72 h, CFSE dye intensity was measured by flow cytometry. The percentage of live cells that have undergone the indicated number of cell divisions is shown. Data are representative of three repeat experiments.

    Journal: PLoS ONE

    Article Title: The FERM and PDZ Domain-Containing Protein Tyrosine Phosphatases, PTPN4 and PTPN3, Are Both Dispensable for T Cell Receptor Signal Transduction

    doi: 10.1371/journal.pone.0004014

    Figure Lengend Snippet: T cell cytokine synthesis and proliferation in PTPN4/PTPN3 double-deficient mice. A) LN T cells were stimulated with the indicated concentrations of CD3 antibody and 0.75 µg/mL CD28 antibody. Concentrations of cytokines in supernatants were determined as in Figure 2 . Each bar represents the mean plus one standard deviation of triplicate determinations from one mouse. Differences between mice are not statistically significant (Student's T-test). Data are representative of six repeat experiments. B) Splenic T cells were labeled with CFSE and stimulated with 0.1 µg/mL CD3 antibody and 0.75 µg/mL CD28 antibody. After 72 h, CFSE dye intensity was measured by flow cytometry. The percentage of live cells that have undergone the indicated number of cell divisions is shown. Data are representative of three repeat experiments.

    Article Snippet: Flow cytometry Thymocytes, splenocytes, and lymph node (LN) cells were blocked with murine IgG (Sigma) and stained with the following conjugated monoclonal antibodies (BD Biosciences): H57-597-APC (TCRβ chain), RA3-6B2-APC-Cy7 (CD45R/B220), IM7-FITC (CD44), GK1.5-APC-Cy7 (CD4), 53-6.7-PerCP (CD8) and PC61-PE (CD25).

    Techniques: Mouse Assay, Standard Deviation, Labeling, Flow Cytometry, Cytometry

    PTPN4/PTPN3 double-deficient mice show normal T cell development. A) Expression of PTPN4 in mice of the indicated genotypes (top) determined by RTPCR. B) Expression of PTPN3 in mice of the indicated genotypes (top) determined by Western blotting. Blots were stripped and reprobed with GAPDH antibodies to confirm equivalent protein loading. C) Flow cytometry plots of thymocytes and peripheral immune cells from PTPN4−/−PTPN3−/−mice and littermate controls showing expression of the indicated markers on live cell populations. In CD44/CD62L plots, the gated population represents recently activated memory T cells. Data are representative of six repeat experiments.

    Journal: PLoS ONE

    Article Title: The FERM and PDZ Domain-Containing Protein Tyrosine Phosphatases, PTPN4 and PTPN3, Are Both Dispensable for T Cell Receptor Signal Transduction

    doi: 10.1371/journal.pone.0004014

    Figure Lengend Snippet: PTPN4/PTPN3 double-deficient mice show normal T cell development. A) Expression of PTPN4 in mice of the indicated genotypes (top) determined by RTPCR. B) Expression of PTPN3 in mice of the indicated genotypes (top) determined by Western blotting. Blots were stripped and reprobed with GAPDH antibodies to confirm equivalent protein loading. C) Flow cytometry plots of thymocytes and peripheral immune cells from PTPN4−/−PTPN3−/−mice and littermate controls showing expression of the indicated markers on live cell populations. In CD44/CD62L plots, the gated population represents recently activated memory T cells. Data are representative of six repeat experiments.

    Article Snippet: Flow cytometry Thymocytes, splenocytes, and lymph node (LN) cells were blocked with murine IgG (Sigma) and stained with the following conjugated monoclonal antibodies (BD Biosciences): H57-597-APC (TCRβ chain), RA3-6B2-APC-Cy7 (CD45R/B220), IM7-FITC (CD44), GK1.5-APC-Cy7 (CD4), 53-6.7-PerCP (CD8) and PC61-PE (CD25).

    Techniques: Mouse Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Flow Cytometry, Cytometry

    Normal T cell development and function in PTPN4-deficient mice. A) Flow cytometry plots of thymocytes, LN cells, and splenocytes from PTPN4-deficient mice and littermate controls showing expression of the indicated markers on live cell populations. Percentages of cells that fall within the indicated regions are shown. Data are representative of three repeat experiments. B) LN T cells were stimulated with the indicated concentrations of CD3 antibody and 0.5 µg/mL CD28 antibody. Concentrations of cytokines in supernatants were determined by ELISA. Each symbol represents the mean of triplicate determinations from a single mouse. Bars represent the mean cytokine secretion. Differences between PTPN4-deficient and control mice are not statistically significant (Paired Student's T test).

    Journal: PLoS ONE

    Article Title: The FERM and PDZ Domain-Containing Protein Tyrosine Phosphatases, PTPN4 and PTPN3, Are Both Dispensable for T Cell Receptor Signal Transduction

    doi: 10.1371/journal.pone.0004014

    Figure Lengend Snippet: Normal T cell development and function in PTPN4-deficient mice. A) Flow cytometry plots of thymocytes, LN cells, and splenocytes from PTPN4-deficient mice and littermate controls showing expression of the indicated markers on live cell populations. Percentages of cells that fall within the indicated regions are shown. Data are representative of three repeat experiments. B) LN T cells were stimulated with the indicated concentrations of CD3 antibody and 0.5 µg/mL CD28 antibody. Concentrations of cytokines in supernatants were determined by ELISA. Each symbol represents the mean of triplicate determinations from a single mouse. Bars represent the mean cytokine secretion. Differences between PTPN4-deficient and control mice are not statistically significant (Paired Student's T test).

    Article Snippet: Flow cytometry Thymocytes, splenocytes, and lymph node (LN) cells were blocked with murine IgG (Sigma) and stained with the following conjugated monoclonal antibodies (BD Biosciences): H57-597-APC (TCRβ chain), RA3-6B2-APC-Cy7 (CD45R/B220), IM7-FITC (CD44), GK1.5-APC-Cy7 (CD4), 53-6.7-PerCP (CD8) and PC61-PE (CD25).

    Techniques: Mouse Assay, Flow Cytometry, Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

    Normal T cell development in PTPN4/PTPN3 double-deficient PTPN13 ΔPTP/ΔPTP mice. Thymocytes and splenocytes from mice of the indicated genotypes were analyzed by flow cytometry for expression of T cell and B cell markers. Shown is the mean percentage representation plus one standard deviation of the indicated subpopulations among total live cells (n = 4 mice of each genotype). Differences between mice are not statistically significant.

    Journal: PLoS ONE

    Article Title: The FERM and PDZ Domain-Containing Protein Tyrosine Phosphatases, PTPN4 and PTPN3, Are Both Dispensable for T Cell Receptor Signal Transduction

    doi: 10.1371/journal.pone.0004014

    Figure Lengend Snippet: Normal T cell development in PTPN4/PTPN3 double-deficient PTPN13 ΔPTP/ΔPTP mice. Thymocytes and splenocytes from mice of the indicated genotypes were analyzed by flow cytometry for expression of T cell and B cell markers. Shown is the mean percentage representation plus one standard deviation of the indicated subpopulations among total live cells (n = 4 mice of each genotype). Differences between mice are not statistically significant.

    Article Snippet: Flow cytometry Thymocytes, splenocytes, and lymph node (LN) cells were blocked with murine IgG (Sigma) and stained with the following conjugated monoclonal antibodies (BD Biosciences): H57-597-APC (TCRβ chain), RA3-6B2-APC-Cy7 (CD45R/B220), IM7-FITC (CD44), GK1.5-APC-Cy7 (CD4), 53-6.7-PerCP (CD8) and PC61-PE (CD25).

    Techniques: Mouse Assay, Flow Cytometry, Cytometry, Expressing, Standard Deviation

    Function of PTPN4/PTPN3 double-deficient PTPN13 ΔPTP/ΔPTP T cells. A) LN T cells were stimulated with the indicated concentrations of CD3 antibody and 0.5 µg/mL CD28 antibody. Concentrations of cytokines in supernatants were determined as in Figure 2 . Differences between mice are not statistically significant, excepting IFN-γ secretion at low dose anti-CD3 which is not a reproducible finding over four repeat experiments. B) Splenic T cells were labeled with CFSE and stimulated with 0.1 µg/mL CD3 antibody and 0.5 µg/mL CD28 antibody. After 72 h, CFSE dye intensity was measured by flow cytometry. Data are representative of four mice of each genotype.

    Journal: PLoS ONE

    Article Title: The FERM and PDZ Domain-Containing Protein Tyrosine Phosphatases, PTPN4 and PTPN3, Are Both Dispensable for T Cell Receptor Signal Transduction

    doi: 10.1371/journal.pone.0004014

    Figure Lengend Snippet: Function of PTPN4/PTPN3 double-deficient PTPN13 ΔPTP/ΔPTP T cells. A) LN T cells were stimulated with the indicated concentrations of CD3 antibody and 0.5 µg/mL CD28 antibody. Concentrations of cytokines in supernatants were determined as in Figure 2 . Differences between mice are not statistically significant, excepting IFN-γ secretion at low dose anti-CD3 which is not a reproducible finding over four repeat experiments. B) Splenic T cells were labeled with CFSE and stimulated with 0.1 µg/mL CD3 antibody and 0.5 µg/mL CD28 antibody. After 72 h, CFSE dye intensity was measured by flow cytometry. Data are representative of four mice of each genotype.

    Article Snippet: Flow cytometry Thymocytes, splenocytes, and lymph node (LN) cells were blocked with murine IgG (Sigma) and stained with the following conjugated monoclonal antibodies (BD Biosciences): H57-597-APC (TCRβ chain), RA3-6B2-APC-Cy7 (CD45R/B220), IM7-FITC (CD44), GK1.5-APC-Cy7 (CD4), 53-6.7-PerCP (CD8) and PC61-PE (CD25).

    Techniques: Mouse Assay, Labeling, Flow Cytometry, Cytometry

    Involvement of ROS in olaparib-mediated cytotoxicity. A. Upper panel, representative flow cytometry histograms to illustrate the olaparib-induced shift of ROS levels as detected by the DCF probe in 639-V cells. A 1 hour treatment with 100 μM H 2 O 2 was used as a positive control. Lower panel, percentage of cells with high ROS levels following olaparib treatment, correct for endogenous ROS levels in the absence of drug. B. Upper panel, representative images illustrating olaparib-induced comets in 639-V cells following 5 hours (h) of drug treatment. Lower panel, quantification of alkaline Comet assay with tail moment plotted against treatments as indicated. Scatter plots show individual data points from 3 independent repeat experiments, with horizontal lines indicating median values. C. Upper panel, DSB kinetics using γ-H2AX and 53BP1 foci formation in olaparib-treated 639-V cells with readouts normalized to untreated controls. Lower panel, parallel determination of ROS formation. D . Fraction of cells after treatment with the anti-oxidant N-acetyl cysteine (NAC) or olaparib was determined by the syto60 assay. The combined drug effect was corrected for the effect of NAC alone as indicated by “n”. NAC was added to cells every 24 hours at 0.5 mM (KU1919, 639-V) or 0.2 mM (5637, UMUC3). For data presentation and statistical comparisons see Fig. 1 , except in Fig. 2B statistical comparison was performed using the Mann-Whitney test.

    Journal: Oncogene

    Article Title: PARP-1 inhibition with or without ionizing radiation confers reactive oxygen species-mediated cytotoxicity preferentially to cancer cells with mutant TP53

    doi: 10.1038/s41388-018-0130-6

    Figure Lengend Snippet: Involvement of ROS in olaparib-mediated cytotoxicity. A. Upper panel, representative flow cytometry histograms to illustrate the olaparib-induced shift of ROS levels as detected by the DCF probe in 639-V cells. A 1 hour treatment with 100 μM H 2 O 2 was used as a positive control. Lower panel, percentage of cells with high ROS levels following olaparib treatment, correct for endogenous ROS levels in the absence of drug. B. Upper panel, representative images illustrating olaparib-induced comets in 639-V cells following 5 hours (h) of drug treatment. Lower panel, quantification of alkaline Comet assay with tail moment plotted against treatments as indicated. Scatter plots show individual data points from 3 independent repeat experiments, with horizontal lines indicating median values. C. Upper panel, DSB kinetics using γ-H2AX and 53BP1 foci formation in olaparib-treated 639-V cells with readouts normalized to untreated controls. Lower panel, parallel determination of ROS formation. D . Fraction of cells after treatment with the anti-oxidant N-acetyl cysteine (NAC) or olaparib was determined by the syto60 assay. The combined drug effect was corrected for the effect of NAC alone as indicated by “n”. NAC was added to cells every 24 hours at 0.5 mM (KU1919, 639-V) or 0.2 mM (5637, UMUC3). For data presentation and statistical comparisons see Fig. 1 , except in Fig. 2B statistical comparison was performed using the Mann-Whitney test.

    Article Snippet: Flow cytometry assays Cell cycle distributions were investigated by standard flow cytometry analysis of DNA staining with propidium iodide (PI) (Sigma-Aldrich).

    Techniques: Flow Cytometry, Cytometry, Positive Control, Alkaline Single Cell Gel Electrophoresis, MANN-WHITNEY

    Effect of ROS on DNA damage and radiosensitization. A. Fraction of cells with high γ-H2AX fluorescence after 5 hours (h) of treatment of olaparib. Fixed cells were co-stained with γ-H2AX and propidium iodide and analyzed by flow cytometry. Cells were divided into G1 phase or S/G2/M phase subgroups for quantifying γ-H2AX fluorescence. B. Percentage of cells with at least 20 53BP1 foci following 48 h of serum starvation +/- 5h olaparib, which led to an enrichment of the cell population with G1 phase cells. C. Representative images showing olaparib-induced 53BP1foci (green) in PCNA (red) negative cells in a MIBC explant. Arrows point to PCNA-negative cells harboring DSB, presumed to be in G0 or G1 phase. D. Sensitivity of 639-V and KU-19-19 cells to the catalytic non-trapping PARP inhibitor ABT-888 as measured by a 6-day cell proliferation assay (syto60). E. Left panel, Representative flow cytometry image showing MitoSOX fluorescence in 639-V cells under different conditions. Right panel, Kinetics of mitochondrial ROS as measured with the MitoSOX probe. F. Effect of a mitochondrial ROS scavenger (MitoTEMPO, 1 μM) on olaparib-induced ROS production in a MIBC explant analogous to Fig. 3D . G. Cell survival fraction of olaparib-treated 639-V cells with or without MitoTEMPO as measured by the syto60 assay. H. Radiosensitization factors (SRF 2Gy ) for representative olaparib-treated bladder cancer cell lines with or without MitoTEMPO or NAC co-incubation. For data presentation and statistical comparisons see Fig. 1 .

    Journal: Oncogene

    Article Title: PARP-1 inhibition with or without ionizing radiation confers reactive oxygen species-mediated cytotoxicity preferentially to cancer cells with mutant TP53

    doi: 10.1038/s41388-018-0130-6

    Figure Lengend Snippet: Effect of ROS on DNA damage and radiosensitization. A. Fraction of cells with high γ-H2AX fluorescence after 5 hours (h) of treatment of olaparib. Fixed cells were co-stained with γ-H2AX and propidium iodide and analyzed by flow cytometry. Cells were divided into G1 phase or S/G2/M phase subgroups for quantifying γ-H2AX fluorescence. B. Percentage of cells with at least 20 53BP1 foci following 48 h of serum starvation +/- 5h olaparib, which led to an enrichment of the cell population with G1 phase cells. C. Representative images showing olaparib-induced 53BP1foci (green) in PCNA (red) negative cells in a MIBC explant. Arrows point to PCNA-negative cells harboring DSB, presumed to be in G0 or G1 phase. D. Sensitivity of 639-V and KU-19-19 cells to the catalytic non-trapping PARP inhibitor ABT-888 as measured by a 6-day cell proliferation assay (syto60). E. Left panel, Representative flow cytometry image showing MitoSOX fluorescence in 639-V cells under different conditions. Right panel, Kinetics of mitochondrial ROS as measured with the MitoSOX probe. F. Effect of a mitochondrial ROS scavenger (MitoTEMPO, 1 μM) on olaparib-induced ROS production in a MIBC explant analogous to Fig. 3D . G. Cell survival fraction of olaparib-treated 639-V cells with or without MitoTEMPO as measured by the syto60 assay. H. Radiosensitization factors (SRF 2Gy ) for representative olaparib-treated bladder cancer cell lines with or without MitoTEMPO or NAC co-incubation. For data presentation and statistical comparisons see Fig. 1 .

    Article Snippet: Flow cytometry assays Cell cycle distributions were investigated by standard flow cytometry analysis of DNA staining with propidium iodide (PI) (Sigma-Aldrich).

    Techniques: Fluorescence, Staining, Flow Cytometry, Cytometry, Proliferation Assay, Incubation

    Flow cytometry analysis showing (a) the percentage of Cy5 positive cells and (b) the mean fluorescent intensity of Cy5 positive cells following a 6 hour incubation of 16HBE14o - cells with Cy5 labelled pCI-Luc complexed into LD (0.75:1), LD (4:1), PD and LPD complexes. Values are mean ± SEM; n = 6; NS, not significant; ***P

    Journal: Scientific Reports

    Article Title: Role of liposome and peptide in the synergistic enhancement of transfection with a lipopolyplex vector

    doi: 10.1038/srep09292

    Figure Lengend Snippet: Flow cytometry analysis showing (a) the percentage of Cy5 positive cells and (b) the mean fluorescent intensity of Cy5 positive cells following a 6 hour incubation of 16HBE14o - cells with Cy5 labelled pCI-Luc complexed into LD (0.75:1), LD (4:1), PD and LPD complexes. Values are mean ± SEM; n = 6; NS, not significant; ***P

    Article Snippet: Flow cytometry Adherent cells in 96 well plates were detached using 50 μL Trypsin-EDTA (Sigma-Aldrich, Dorset, UK) and re-suspended with 150 μL DPBS (Sigma-Aldrich, Dorset, UK).

    Techniques: Flow Cytometry, Cytometry, Incubation

    Flow cytometry analyses of BMSCs concentrations in peripheral blood CD34−/CD29 + and CXCR4 + are surface markers of BMSC and the graphs show the concentration of BMSCs in peripheral blood. CD29 was labeled with PE/Cy5, CD34 with PE and CXCR4 with Alexa Fluor ® 488. Specific values of CD34−/CD29 + cells and CXCR4 + cells were marked in the corresponding position of the squares. The concentration of BMSCs in peripheral blood significantly increased after transfection with SDF-1α genes. ( n = 10).

    Journal: Oncotarget

    Article Title: Homing of endogenous bone marrow mesenchymal stem cells to rat infarcted myocardium via ultrasound-mediated recombinant SDF-1α adenovirus in microbubbles

    doi: 10.18632/oncotarget.23068

    Figure Lengend Snippet: Flow cytometry analyses of BMSCs concentrations in peripheral blood CD34−/CD29 + and CXCR4 + are surface markers of BMSC and the graphs show the concentration of BMSCs in peripheral blood. CD29 was labeled with PE/Cy5, CD34 with PE and CXCR4 with Alexa Fluor ® 488. Specific values of CD34−/CD29 + cells and CXCR4 + cells were marked in the corresponding position of the squares. The concentration of BMSCs in peripheral blood significantly increased after transfection with SDF-1α genes. ( n = 10).

    Article Snippet: Flow cytometry Flow cytometry (FACS Diva Version 6.1.3 US Becton Dickinson Company) was used to detect BMSC surface markers CD34−CD29 + and CXCR4 +, and the amount of BMSCs in peripheral blood.

    Techniques: Flow Cytometry, Cytometry, Concentration Assay, Labeling, Transfection

    Flow cytometry detection of apoptosis. dsRNA induced HepG2.2.15 cell apoptosis ( ∗ P

    Journal: Gastroenterology Research and Practice

    Article Title: TLR3 Plays Significant Roles against HBV-Associated HCC

    doi: 10.1155/2015/572171

    Figure Lengend Snippet: Flow cytometry detection of apoptosis. dsRNA induced HepG2.2.15 cell apoptosis ( ∗ P

    Article Snippet: Flow Cytometry Assay Flow cytometry (Beckman Coulter, Fullerton, CA, USA) was used to determine the apoptotic rate.

    Techniques: Flow Cytometry, Cytometry