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  • 99
    Millipore flow cytometry
    Characterization of microparticles generated by stimulated HUT-78 cells (MP T ). (A) Scanning electron microscopy of isolated MP T . Scale bar = 500 nm. (B) Flow <t>cytometry</t> analysis of the binding of FITC-annexin V to MP T . (C) Monocytes (5×10 4 cells/200 µl/well; 96-well plates) were activated by MP T (3 µg/ml) for 24 h in the presence (empty columns) or absence (grey columns) of HDL (0.2 mg/ml proteins). Cell culture supernatants were measured for the presence of the indicated cytokines. Results are expressed as mean ± SD of 3 experiments carried out with monocytes isolated from 3 individual donors. ** p
    Flow Cytometry, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6390 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    flow cytometry - by Bioz Stars, 2020-08
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    99
    BioLegend flow cytometry flow cytometry
    BTNL3 Binding to Vγ4 Involves Germline-Encoded Regions, whereas Antigen-Specific Binding Requires CDR3γ and CDR3δ Regions (A) Amino acid sequence of human Vγ4 and Vδ5 from the LES clone, showing mutations tested in CDR1, CDR2, HV4, and CDR3 in red below (top). (Middle) Amino acid sequence of human Vγ4 from the hu17 TCR aligned to human Vγ3 and the indicated Vγ3/hu17 Vγ4 hybrids transduced in JRT3. Red font indicates divergence from WT Vγ4. (Bottom) Alignment of the amino acid sequences of human Vγ4 with mouse Vγ7 and the CDR2 and HV4 chimeras generated in the mo5 Vγ7Vδ2-2 TCR. Red font indicates amino acids from human Vγ4 inserted in mouse Vγ7 to generate the indicated chimeras, expressed in Jurkat 76. (B) Binding affinity of BTNL3 to indicated Vγ4 and Vδ5 mutants (mutant K d ) relative to WT LES TCR affinity (WT K d ) measured in the same experiment. The averages of n = 4–5 experiments per Vγ4 mutant and 1–2 experiments per Vδ5 mutant are shown. (C) Flow <t>cytometry</t> analysis of TCR downregulation (x axis) plotted against that of CD69 upregulation (y axis) on JRT3 cells transduced with wild-type (WT) Vγ4Vδ1 TCR or the indicated Vγ3 or Vγ4 TCR hybrids and co-cultured for 4 h with 293T.L3L8 cells. Results were normalized to those obtained by co-culture with 293T.EV cells. Data are representative of two independent experiments (mean ± SD of n = 3 co-cultures). (D and E) Flow cytometry analysis of TCR downregulation (D) and CD69 upregulation (E) on J76 cells transduced with mo5 (Vγ7Vδ2-2) TCR or the indicated moVγ7/huVγ4 hybrid TCRs after co-culture with 293T.l1l6 or 293T.L3L8 cells. Results were normalized to those obtained by co-culture with 293T.EV cells. Data are representative of three independent experiments (mean ± SD of n = 3 co-cultures). (F) Binding affinity of EPCR to Vγ4 and Vδ5 mutants (mutant K d ) relative to WT LES TCR affinity (WT K d ) measured in the same experiment, representative of 2–3 experiments. (G) EPCR (3,012 RU) or control protein (2,586 RU) were immobilized on the sensor chip. WT LES TCR was injected over the surface at 12.5 μM in the presence of increasing specific competitor (BTNL3 IgV) or non-specific competitor (BTNL8 IgV). Binding responses were measured and are shown as a percentage of binding observed in the absence of competitor. See also Figure S3 .
    Flow Cytometry Flow Cytometry, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc flow cytometry
    BTNL3 Binding to Vγ4 Involves Germline-Encoded Regions, whereas Antigen-Specific Binding Requires CDR3γ and CDR3δ Regions (A) Amino acid sequence of human Vγ4 and Vδ5 from the LES clone, showing mutations tested in CDR1, CDR2, HV4, and CDR3 in red below (top). (Middle) Amino acid sequence of human Vγ4 from the hu17 TCR aligned to human Vγ3 and the indicated Vγ3/hu17 Vγ4 hybrids transduced in JRT3. Red font indicates divergence from WT Vγ4. (Bottom) Alignment of the amino acid sequences of human Vγ4 with mouse Vγ7 and the CDR2 and HV4 chimeras generated in the mo5 Vγ7Vδ2-2 TCR. Red font indicates amino acids from human Vγ4 inserted in mouse Vγ7 to generate the indicated chimeras, expressed in Jurkat 76. (B) Binding affinity of BTNL3 to indicated Vγ4 and Vδ5 mutants (mutant K d ) relative to WT LES TCR affinity (WT K d ) measured in the same experiment. The averages of n = 4–5 experiments per Vγ4 mutant and 1–2 experiments per Vδ5 mutant are shown. (C) Flow <t>cytometry</t> analysis of TCR downregulation (x axis) plotted against that of CD69 upregulation (y axis) on JRT3 cells transduced with wild-type (WT) Vγ4Vδ1 TCR or the indicated Vγ3 or Vγ4 TCR hybrids and co-cultured for 4 h with 293T.L3L8 cells. Results were normalized to those obtained by co-culture with 293T.EV cells. Data are representative of two independent experiments (mean ± SD of n = 3 co-cultures). (D and E) Flow cytometry analysis of TCR downregulation (D) and CD69 upregulation (E) on J76 cells transduced with mo5 (Vγ7Vδ2-2) TCR or the indicated moVγ7/huVγ4 hybrid TCRs after co-culture with 293T.l1l6 or 293T.L3L8 cells. Results were normalized to those obtained by co-culture with 293T.EV cells. Data are representative of three independent experiments (mean ± SD of n = 3 co-cultures). (F) Binding affinity of EPCR to Vγ4 and Vδ5 mutants (mutant K d ) relative to WT LES TCR affinity (WT K d ) measured in the same experiment, representative of 2–3 experiments. (G) EPCR (3,012 RU) or control protein (2,586 RU) were immobilized on the sensor chip. WT LES TCR was injected over the surface at 12.5 μM in the presence of increasing specific competitor (BTNL3 IgV) or non-specific competitor (BTNL8 IgV). Binding responses were measured and are shown as a percentage of binding observed in the absence of competitor. See also Figure S3 .
    Flow Cytometry, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 300 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson flow cytometry buffer
    The level of IRF4 phosphorylation correlates with LMP1 and is dampened by the Src-specific inhibitor PP2 in EBV + cells. ( a ) Cells were collected, washed and fixed after treatment with 15 μM PP2 or DMSO (control) for 24 h. Cells were then permeabilized, and incubated with mouse anti-LMP1 antibody (Dako) and rabbit anti-p-IRF4 antibody (21st Century Biochemicals). After wash, cells were incubated with anti-mouse IgG APC (eBioscience) and anti-rabbit PE (eBioscience) before subjected to flow <t>cytometry</t> analysis. Q4 represents the portion of LMP1 + /p-IRF4 + cells. ( b ) SavIII cells were transfected with p85 shRNA (#1) (or control). shRNA expression was induced by 1 μg/ml doxycycline for 3 days. p-IRF4 was then evaluated by flow. P = 0.0007 (unpaired t -test). ( c ) BJAB cells were transfected with IRF4, IRF4 plus LMP1 (or c-Src as a positive control). Cells were subjected to flow analysis after 48 h. Statistical analysis was performed for three independent flow analyses. Results are the averages ± s.d. Representative results from at least three independent experiments are shown.
    Flow Cytometry Buffer, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 1435 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    R&D Systems flow cytometry buffer
    Flow <t>cytometry</t> analysis of cell surface marker expression. (A) Expression of surface markers CD14, CD19, CD34, CD45, HLA-DR, CD73, CD90, CD105 (filled) with relevant isotype control (unfilled). (B) Quantification of the percentage of positive events compared to the relevant isotype control. Markers set to exclude 99% of isotype control events; ≥ 4x10 4 events were collected per sample. OK3 was tested at PD10, OK3H at PD50, BMA13 at PD6, BMA13H at PD18, 1C6 at PD44 and 1C6H at PD118.
    Flow Cytometry Buffer, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    PAA Laboratories flow cytometry buffer
    Characterization of gp130-dNG expression and function in stably transduced BAF3 cell pools. A , flow <t>cytometry</t> analysis of total gp130-EYFP and gp130-dNG-EYFP expression (fluorescein isothiocyanate/GFP channel; black ) versus the nonspecific signal of untransduced
    Flow Cytometry Buffer, supplied by PAA Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher flow cytometry buffer
    Lung cells infected by CAL- and HA mut viruses. Five Balb/c female mice/condition of 6–9 weeks of age were infected with 10 3 pfu of CAL and HA-mut viruses or mock infected and pooled into one sample. At days 1, 2, and 3 post-infection lungs were collected, and processed for flow <t>cytometry.</t> Single cell suspension was stained with antibodies against NP, EpCam and CD45, to detect infected cells, epithelial cells and leukocytes, respectively. (A) Shows events positive for NP in the y-axis. (B,C) Percentage of specific populations of infected cells in relation to the total events, total number of gated EpCam or of CD45 positive cells were plotted. (D) Total percentage of CD45 positive cells, regardless of the infectivity state were plotted. The experiment was performed twice and overall 5 mice per condition pooled into one sample. (E,F) Mice ( n = 5) were inoculated intranasally with a sublethal dose (10 3 pfu) of each recombinant virus or PBS (MOCK) as control and were evaluated in three groups. Neutrophils (E) and alveolar macrophages (F) were quantified in the lungs (% of cells) at indicated dpi. Significance was determined by Student's t -test ( * p
    Flow Cytometry Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 724 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Santa Cruz Biotechnology flow cytometry buffer
    Silencing of Gal3 shifts CSC subset to a Gal3 negative CSC subset. ( a ) Western blot analysis for comparison of total Gal3 in LiM6TR or DLD1TR colon cancer cells and L3.6pl or AsPC1 pancreatic cancer cells after infection with lentiviral particles for control-shRNA ( c ) or Gal3-shRNA (G). Each pair of control and Gal3 knockdown cells were run side by side on the same gel. Comparison of Gal3 expression between cell lines has been previously published 10 ( b ) Flow cytometric analysis of colon cancer and pancreatic cancer cells after infection with lentiviral particles for control-shRNA (red trace) or Gal3-shRNA (green trace). Black trace is background staining. ( c ) ALDH activity was analyzed by flow <t>cytometry.</t> ALDEFLUOR activity in colon cancer and pancreatic cancer cells after infection with lentiviral particles for control-shRNA (blue trace) or Gal3-shRNA (green trace). Gray profile is background staining
    Flow Cytometry Buffer, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Streck Laboratories flow cytometry buffer
    Silencing of Gal3 shifts CSC subset to a Gal3 negative CSC subset. ( a ) Western blot analysis for comparison of total Gal3 in LiM6TR or DLD1TR colon cancer cells and L3.6pl or AsPC1 pancreatic cancer cells after infection with lentiviral particles for control-shRNA ( c ) or Gal3-shRNA (G). Each pair of control and Gal3 knockdown cells were run side by side on the same gel. Comparison of Gal3 expression between cell lines has been previously published 10 ( b ) Flow cytometric analysis of colon cancer and pancreatic cancer cells after infection with lentiviral particles for control-shRNA (red trace) or Gal3-shRNA (green trace). Black trace is background staining. ( c ) ALDH activity was analyzed by flow <t>cytometry.</t> ALDEFLUOR activity in colon cancer and pancreatic cancer cells after infection with lentiviral particles for control-shRNA (blue trace) or Gal3-shRNA (green trace). Gray profile is background staining
    Flow Cytometry Buffer, supplied by Streck Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    R&D Systems flow cytometry fixation buffer
    Estrogen abrogates HCV infection. (A) Huh7.5 cells respond to estrogen treatment measured by cyclin-D1 expression. (B) HCV core protein in J6/JFH1-infected Huh7.5 cells treated with E2 or Universal Type I IFN. (C) HCV intracellular core protein in J6/JFH1-infected Huh7.5 cells treated with E2, as measured by Flow <t>Cytometry.</t> (D) HCV RNA quantitated relative to GAPDH RNA levels from infected Huh7.5 cells treated with E2 or IFN. (E) Virus growth measured by absolute mean focus forming units (FFU). Error bars indicate the statistical standard deviation from the mean (±SD). Statistical significance is indicated by asterisks where: (*** = P ≤ 0.001; **** = P ≤ 0.0001). (F) Representative images of foci after 48 hr of E2 treatment. Magnification 40X ( below ); 100X ( above ).
    Flow Cytometry Fixation Buffer, supplied by R&D Systems, used in various techniques. Bioz Stars score: 88/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore flow cytometry buffer
    Spore-FP1 induces enrichment of tissue resident memory cells. Mice were first immunized with Bacillus Calmette-Guérin for 6 weeks (except the PBS group) and then received two intranasal doses of either spores alone, fusion protein 1 (FP1) alone, or Spore-FP1. Lung parenchymal cells were assessed by flow <t>cytometry</t> for T-cell markers. A gating strategy of live cells→single cells→CD3 + →CD4 + /CD8 + →CD44 hi CD62L lo was used to measure the frequency of double-positive CD69/CD103 Trm. Data are derived from n = 3 pooled mice per group showing a representative plot.
    Flow Cytometry Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 426 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    R&D Systems flow cytometry permeabilization buffer
    Spore-FP1 induces enrichment of tissue resident memory cells. Mice were first immunized with Bacillus Calmette-Guérin for 6 weeks (except the PBS group) and then received two intranasal doses of either spores alone, fusion protein 1 (FP1) alone, or Spore-FP1. Lung parenchymal cells were assessed by flow <t>cytometry</t> for T-cell markers. A gating strategy of live cells→single cells→CD3 + →CD4 + /CD8 + →CD44 hi CD62L lo was used to measure the frequency of double-positive CD69/CD103 Trm. Data are derived from n = 3 pooled mice per group showing a representative plot.
    Flow Cytometry Permeabilization Buffer, supplied by R&D Systems, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Tonbo Biosciences flow cytometry perm buffer
    Enhanced ZIKV infection of HCs is modulated in an IgG subclass-dependent manner. A) Flow <t>cytometry</t> plots showing expression of FcγRIII (CD16), FcγRII (CD32), and FcγRI (CD64) of uninfected HCs. Representative experiment from n=2
    Flow Cytometry Perm Buffer, supplied by Tonbo Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Novus Biologicals flow cytometry
    Enhanced ZIKV infection of HCs is modulated in an IgG subclass-dependent manner. A) Flow <t>cytometry</t> plots showing expression of FcγRIII (CD16), FcγRII (CD32), and FcγRI (CD64) of uninfected HCs. Representative experiment from n=2
    Flow Cytometry, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Becton Dickinson polychromatic flow cytometry
    Enhanced ZIKV infection of HCs is modulated in an IgG subclass-dependent manner. A) Flow <t>cytometry</t> plots showing expression of FcγRIII (CD16), FcγRII (CD32), and FcγRI (CD64) of uninfected HCs. Representative experiment from n=2
    Polychromatic Flow Cytometry, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 134 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Characterization of microparticles generated by stimulated HUT-78 cells (MP T ). (A) Scanning electron microscopy of isolated MP T . Scale bar = 500 nm. (B) Flow cytometry analysis of the binding of FITC-annexin V to MP T . (C) Monocytes (5×10 4 cells/200 µl/well; 96-well plates) were activated by MP T (3 µg/ml) for 24 h in the presence (empty columns) or absence (grey columns) of HDL (0.2 mg/ml proteins). Cell culture supernatants were measured for the presence of the indicated cytokines. Results are expressed as mean ± SD of 3 experiments carried out with monocytes isolated from 3 individual donors. ** p

    Journal: PLoS ONE

    Article Title: HDL Interfere with the Binding of T Cell Microparticles to Human Monocytes to Inhibit Pro-Inflammatory Cytokine Production

    doi: 10.1371/journal.pone.0011869

    Figure Lengend Snippet: Characterization of microparticles generated by stimulated HUT-78 cells (MP T ). (A) Scanning electron microscopy of isolated MP T . Scale bar = 500 nm. (B) Flow cytometry analysis of the binding of FITC-annexin V to MP T . (C) Monocytes (5×10 4 cells/200 µl/well; 96-well plates) were activated by MP T (3 µg/ml) for 24 h in the presence (empty columns) or absence (grey columns) of HDL (0.2 mg/ml proteins). Cell culture supernatants were measured for the presence of the indicated cytokines. Results are expressed as mean ± SD of 3 experiments carried out with monocytes isolated from 3 individual donors. ** p

    Article Snippet: Buffers used for flow cytometry analysis were subjected to filtration (Stericup 0.22 µm, Millipore) to discard interferences with small debris.

    Techniques: Generated, Electron Microscopy, Isolation, Flow Cytometry, Cytometry, Binding Assay, Cell Culture

    HDL inhibit the binding of MP T to human monocytes. The binding of PKH67-labelled MP T to CD14 + monocytes in the presence or absence of HDL was measured by flow cytometry. (A) Representative binding of PKH67-labelled MP T (12 µg/ml proteins) to CD14 + monocytes in the presence or absence of 0.2 mg/ml HDL (as indicated). (B) Flow cytometry measurement of the binding of increasing concentration of PKH67-labelled MP T to CD14 + monocytes in the absence (closed circles) or presence (empty circles) of 0.2 mg/ml HDL. The percentage ± SD of MP T + CD14 + monocytes (upper right panel) in 3 different experiments is presented.

    Journal: PLoS ONE

    Article Title: HDL Interfere with the Binding of T Cell Microparticles to Human Monocytes to Inhibit Pro-Inflammatory Cytokine Production

    doi: 10.1371/journal.pone.0011869

    Figure Lengend Snippet: HDL inhibit the binding of MP T to human monocytes. The binding of PKH67-labelled MP T to CD14 + monocytes in the presence or absence of HDL was measured by flow cytometry. (A) Representative binding of PKH67-labelled MP T (12 µg/ml proteins) to CD14 + monocytes in the presence or absence of 0.2 mg/ml HDL (as indicated). (B) Flow cytometry measurement of the binding of increasing concentration of PKH67-labelled MP T to CD14 + monocytes in the absence (closed circles) or presence (empty circles) of 0.2 mg/ml HDL. The percentage ± SD of MP T + CD14 + monocytes (upper right panel) in 3 different experiments is presented.

    Article Snippet: Buffers used for flow cytometry analysis were subjected to filtration (Stericup 0.22 µm, Millipore) to discard interferences with small debris.

    Techniques: Binding Assay, Flow Cytometry, Cytometry, Concentration Assay

    MP T specifically bind and activate human monocytes. (A–C) The binding of PKH67-labelled MP from different cellular sources to isolated human monocytes and T lymphocytes was assessed by flow cytometry. Binding of MP T (12 µg/ml) to CD14 + monocytes (A) and CD3 + T lymphocytes (B). (C) Binding of endothelial cell MP (MP EC ; 12 µg/ml) to CD14 + monocytes. (D) Monocytes (5×10 4 cells/well/200 µl/well; 96-well plates) were activated by 3 µg/ml MP T , 14 µg/ml activated endothelial cells (MP EC ) and 14 µg/ml activated platelets (PMP) in the presence (empty columns) or absence (grey columns) of 0.2 mg/ml HDL. IL-1β was measured in culture supernatants after 24 h incubation. Results are expressed as mean ± SD of triplicates.

    Journal: PLoS ONE

    Article Title: HDL Interfere with the Binding of T Cell Microparticles to Human Monocytes to Inhibit Pro-Inflammatory Cytokine Production

    doi: 10.1371/journal.pone.0011869

    Figure Lengend Snippet: MP T specifically bind and activate human monocytes. (A–C) The binding of PKH67-labelled MP from different cellular sources to isolated human monocytes and T lymphocytes was assessed by flow cytometry. Binding of MP T (12 µg/ml) to CD14 + monocytes (A) and CD3 + T lymphocytes (B). (C) Binding of endothelial cell MP (MP EC ; 12 µg/ml) to CD14 + monocytes. (D) Monocytes (5×10 4 cells/well/200 µl/well; 96-well plates) were activated by 3 µg/ml MP T , 14 µg/ml activated endothelial cells (MP EC ) and 14 µg/ml activated platelets (PMP) in the presence (empty columns) or absence (grey columns) of 0.2 mg/ml HDL. IL-1β was measured in culture supernatants after 24 h incubation. Results are expressed as mean ± SD of triplicates.

    Article Snippet: Buffers used for flow cytometry analysis were subjected to filtration (Stericup 0.22 µm, Millipore) to discard interferences with small debris.

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

    HDL interaction with MP T . The binding of FITC-HDL to monocytes (A), MP T (B) and MP from unstimulated HUT-78 cells (C) was analyzed by flow cytometry. Results are representative of 3 different experiments.

    Journal: PLoS ONE

    Article Title: HDL Interfere with the Binding of T Cell Microparticles to Human Monocytes to Inhibit Pro-Inflammatory Cytokine Production

    doi: 10.1371/journal.pone.0011869

    Figure Lengend Snippet: HDL interaction with MP T . The binding of FITC-HDL to monocytes (A), MP T (B) and MP from unstimulated HUT-78 cells (C) was analyzed by flow cytometry. Results are representative of 3 different experiments.

    Article Snippet: Buffers used for flow cytometry analysis were subjected to filtration (Stericup 0.22 µm, Millipore) to discard interferences with small debris.

    Techniques: Binding Assay, Flow Cytometry, Cytometry

    BTNL3 Binding to Vγ4 Involves Germline-Encoded Regions, whereas Antigen-Specific Binding Requires CDR3γ and CDR3δ Regions (A) Amino acid sequence of human Vγ4 and Vδ5 from the LES clone, showing mutations tested in CDR1, CDR2, HV4, and CDR3 in red below (top). (Middle) Amino acid sequence of human Vγ4 from the hu17 TCR aligned to human Vγ3 and the indicated Vγ3/hu17 Vγ4 hybrids transduced in JRT3. Red font indicates divergence from WT Vγ4. (Bottom) Alignment of the amino acid sequences of human Vγ4 with mouse Vγ7 and the CDR2 and HV4 chimeras generated in the mo5 Vγ7Vδ2-2 TCR. Red font indicates amino acids from human Vγ4 inserted in mouse Vγ7 to generate the indicated chimeras, expressed in Jurkat 76. (B) Binding affinity of BTNL3 to indicated Vγ4 and Vδ5 mutants (mutant K d ) relative to WT LES TCR affinity (WT K d ) measured in the same experiment. The averages of n = 4–5 experiments per Vγ4 mutant and 1–2 experiments per Vδ5 mutant are shown. (C) Flow cytometry analysis of TCR downregulation (x axis) plotted against that of CD69 upregulation (y axis) on JRT3 cells transduced with wild-type (WT) Vγ4Vδ1 TCR or the indicated Vγ3 or Vγ4 TCR hybrids and co-cultured for 4 h with 293T.L3L8 cells. Results were normalized to those obtained by co-culture with 293T.EV cells. Data are representative of two independent experiments (mean ± SD of n = 3 co-cultures). (D and E) Flow cytometry analysis of TCR downregulation (D) and CD69 upregulation (E) on J76 cells transduced with mo5 (Vγ7Vδ2-2) TCR or the indicated moVγ7/huVγ4 hybrid TCRs after co-culture with 293T.l1l6 or 293T.L3L8 cells. Results were normalized to those obtained by co-culture with 293T.EV cells. Data are representative of three independent experiments (mean ± SD of n = 3 co-cultures). (F) Binding affinity of EPCR to Vγ4 and Vδ5 mutants (mutant K d ) relative to WT LES TCR affinity (WT K d ) measured in the same experiment, representative of 2–3 experiments. (G) EPCR (3,012 RU) or control protein (2,586 RU) were immobilized on the sensor chip. WT LES TCR was injected over the surface at 12.5 μM in the presence of increasing specific competitor (BTNL3 IgV) or non-specific competitor (BTNL8 IgV). Binding responses were measured and are shown as a percentage of binding observed in the absence of competitor. See also Figure S3 .

    Journal: Immunity

    Article Title: Butyrophilin-like 3 Directly Binds a Human Vγ4+ T Cell Receptor Using a Modality Distinct from Clonally-Restricted Antigen

    doi: 10.1016/j.immuni.2019.09.006

    Figure Lengend Snippet: BTNL3 Binding to Vγ4 Involves Germline-Encoded Regions, whereas Antigen-Specific Binding Requires CDR3γ and CDR3δ Regions (A) Amino acid sequence of human Vγ4 and Vδ5 from the LES clone, showing mutations tested in CDR1, CDR2, HV4, and CDR3 in red below (top). (Middle) Amino acid sequence of human Vγ4 from the hu17 TCR aligned to human Vγ3 and the indicated Vγ3/hu17 Vγ4 hybrids transduced in JRT3. Red font indicates divergence from WT Vγ4. (Bottom) Alignment of the amino acid sequences of human Vγ4 with mouse Vγ7 and the CDR2 and HV4 chimeras generated in the mo5 Vγ7Vδ2-2 TCR. Red font indicates amino acids from human Vγ4 inserted in mouse Vγ7 to generate the indicated chimeras, expressed in Jurkat 76. (B) Binding affinity of BTNL3 to indicated Vγ4 and Vδ5 mutants (mutant K d ) relative to WT LES TCR affinity (WT K d ) measured in the same experiment. The averages of n = 4–5 experiments per Vγ4 mutant and 1–2 experiments per Vδ5 mutant are shown. (C) Flow cytometry analysis of TCR downregulation (x axis) plotted against that of CD69 upregulation (y axis) on JRT3 cells transduced with wild-type (WT) Vγ4Vδ1 TCR or the indicated Vγ3 or Vγ4 TCR hybrids and co-cultured for 4 h with 293T.L3L8 cells. Results were normalized to those obtained by co-culture with 293T.EV cells. Data are representative of two independent experiments (mean ± SD of n = 3 co-cultures). (D and E) Flow cytometry analysis of TCR downregulation (D) and CD69 upregulation (E) on J76 cells transduced with mo5 (Vγ7Vδ2-2) TCR or the indicated moVγ7/huVγ4 hybrid TCRs after co-culture with 293T.l1l6 or 293T.L3L8 cells. Results were normalized to those obtained by co-culture with 293T.EV cells. Data are representative of three independent experiments (mean ± SD of n = 3 co-cultures). (F) Binding affinity of EPCR to Vγ4 and Vδ5 mutants (mutant K d ) relative to WT LES TCR affinity (WT K d ) measured in the same experiment, representative of 2–3 experiments. (G) EPCR (3,012 RU) or control protein (2,586 RU) were immobilized on the sensor chip. WT LES TCR was injected over the surface at 12.5 μM in the presence of increasing specific competitor (BTNL3 IgV) or non-specific competitor (BTNL8 IgV). Binding responses were measured and are shown as a percentage of binding observed in the absence of competitor. See also Figure S3 .

    Article Snippet: Flow Cytometry Flow cytometry was performed using the following antibodies from BioLegend, unless otherwise stated.

    Techniques: Binding Assay, Sequencing, Generated, Mutagenesis, Flow Cytometry, Cytometry, Transduction, Cell Culture, Co-Culture Assay, Chromatin Immunoprecipitation, Injection

    Regions of BTNL3 IgV Involved in Vγ4 Binding (A) Alignment of BTNL3 and BTNL8 IgV domains showing mutants generated. (B–D) Equilibrium affinity analysis of the binding of (B) BTNL3 GQFSS IgV (K d = 117.6 μM), (C) BTNL3 KDQPFM mutant (K d = 11.2 μM), or (D) BTNL3 RI mutant (K d = 217.3 μM) to Vγ4 TCR. (E) Binding affinity of indicated mutants of BTNL3 (mutant K d ) relative to WT LES TCR affinity (WT K d ) measured in the same experiment. Data are representative of two experiments. (F) Flow cytometry analysis of 293T cells co-transduced with BTNL3 variants (as shown in the legend) and BTNL8 and stained with increasing concentrations (x axis) of soluble Vγ4Vδ1 and anti-His mAb. Results are presented as geometric mean fluorescence intensity (gMFI) of staining with the sTCR plus antibody to the His tag, normalized to the staining of 293T.EV cells under the same conditions. See also Figure S4 .

    Journal: Immunity

    Article Title: Butyrophilin-like 3 Directly Binds a Human Vγ4+ T Cell Receptor Using a Modality Distinct from Clonally-Restricted Antigen

    doi: 10.1016/j.immuni.2019.09.006

    Figure Lengend Snippet: Regions of BTNL3 IgV Involved in Vγ4 Binding (A) Alignment of BTNL3 and BTNL8 IgV domains showing mutants generated. (B–D) Equilibrium affinity analysis of the binding of (B) BTNL3 GQFSS IgV (K d = 117.6 μM), (C) BTNL3 KDQPFM mutant (K d = 11.2 μM), or (D) BTNL3 RI mutant (K d = 217.3 μM) to Vγ4 TCR. (E) Binding affinity of indicated mutants of BTNL3 (mutant K d ) relative to WT LES TCR affinity (WT K d ) measured in the same experiment. Data are representative of two experiments. (F) Flow cytometry analysis of 293T cells co-transduced with BTNL3 variants (as shown in the legend) and BTNL8 and stained with increasing concentrations (x axis) of soluble Vγ4Vδ1 and anti-His mAb. Results are presented as geometric mean fluorescence intensity (gMFI) of staining with the sTCR plus antibody to the His tag, normalized to the staining of 293T.EV cells under the same conditions. See also Figure S4 .

    Article Snippet: Flow Cytometry Flow cytometry was performed using the following antibodies from BioLegend, unless otherwise stated.

    Techniques: Binding Assay, Generated, Mutagenesis, Flow Cytometry, Cytometry, Transduction, Staining, Fluorescence

    The HV4 Region of the Vγ9 TCR and the CFG Face of BTN3A1 and BTN3A2 Are Involved in the Phosphoantigen-Induced Activation of Vγ9Vδ2 T Cells (A) Best fit hypothetical model for docking of the Vγ4 TCR V domain (light gray) onto the BTNL3 IgV domain (green) ( Melandri et al., 2018 ), generated using the computational docking program SwarmDock. Side chains are displayed for amino acids potentially directly involved in the contact between the Vγ4 HV4 region (D, Y, and R; pink) and the CFG face of the BTNL3-IgV domain (H61, W115, and E124; orange, blue, and red, respectively). (B and C) Alignment of the HV4 region of the Vγ4 and Vγ9 TCR V domains (B) and the IgV domains of BTNL3 and BTN3A1 and BTN3A2 (C). Amino acids of interest are colored as in (A). (D) Flow cytometry analysis of the expression of the indicated Vγ9Vδ2 TCR variants by JRT3 cells, 72 h post-transduction. (E) Flow cytometry analysis of CD69 upregulation by JRT3 cells expressing the indicated Vγ9Vδ2 TCR variants, following incubation with media only, or 293T cells with or without pre-treatment with zoledronate (Zol, 10 μM). Data are representative of three experiments (mean ± SD of n = 3 co-cultures). (F) Flow cytometry analysis of CD107a upregulation by polyclonal Vγ9Vδ2 T cell lines derived from peripheral blood mononuclear cells from two donors following co-culture with CRA123 cells transfected with the indicated BTN3A1 constructs (EV, empty vector control) and pre-treated with 10 μM Zol. Data are the mean ± SD of n = 3 co-cultures for each donor. (G) Flow cytometry analysis of CD107a upregulation by a polyclonal Vγ9Vδ2 T cell line following co-culture with CRA123 cells co-transfected with the indicated BTN3A1 + BTN3A2 constructs (EV, empty vector control) and pre-treated with 10 μM Zol. Data are the mean ± SD of n = 3 co-cultures. See also Figure S5 .

    Journal: Immunity

    Article Title: Butyrophilin-like 3 Directly Binds a Human Vγ4+ T Cell Receptor Using a Modality Distinct from Clonally-Restricted Antigen

    doi: 10.1016/j.immuni.2019.09.006

    Figure Lengend Snippet: The HV4 Region of the Vγ9 TCR and the CFG Face of BTN3A1 and BTN3A2 Are Involved in the Phosphoantigen-Induced Activation of Vγ9Vδ2 T Cells (A) Best fit hypothetical model for docking of the Vγ4 TCR V domain (light gray) onto the BTNL3 IgV domain (green) ( Melandri et al., 2018 ), generated using the computational docking program SwarmDock. Side chains are displayed for amino acids potentially directly involved in the contact between the Vγ4 HV4 region (D, Y, and R; pink) and the CFG face of the BTNL3-IgV domain (H61, W115, and E124; orange, blue, and red, respectively). (B and C) Alignment of the HV4 region of the Vγ4 and Vγ9 TCR V domains (B) and the IgV domains of BTNL3 and BTN3A1 and BTN3A2 (C). Amino acids of interest are colored as in (A). (D) Flow cytometry analysis of the expression of the indicated Vγ9Vδ2 TCR variants by JRT3 cells, 72 h post-transduction. (E) Flow cytometry analysis of CD69 upregulation by JRT3 cells expressing the indicated Vγ9Vδ2 TCR variants, following incubation with media only, or 293T cells with or without pre-treatment with zoledronate (Zol, 10 μM). Data are representative of three experiments (mean ± SD of n = 3 co-cultures). (F) Flow cytometry analysis of CD107a upregulation by polyclonal Vγ9Vδ2 T cell lines derived from peripheral blood mononuclear cells from two donors following co-culture with CRA123 cells transfected with the indicated BTN3A1 constructs (EV, empty vector control) and pre-treated with 10 μM Zol. Data are the mean ± SD of n = 3 co-cultures for each donor. (G) Flow cytometry analysis of CD107a upregulation by a polyclonal Vγ9Vδ2 T cell line following co-culture with CRA123 cells co-transfected with the indicated BTN3A1 + BTN3A2 constructs (EV, empty vector control) and pre-treated with 10 μM Zol. Data are the mean ± SD of n = 3 co-cultures. See also Figure S5 .

    Article Snippet: Flow Cytometry Flow cytometry was performed using the following antibodies from BioLegend, unless otherwise stated.

    Techniques: Activation Assay, Generated, Flow Cytometry, Cytometry, Expressing, Transduction, Incubation, Derivative Assay, Co-Culture Assay, Transfection, Construct, Plasmid Preparation

    Mouse Vγ7 TCR-Dependent Recognition of Btnl1.6 (A and B) Flow cytometry analysis of TCR downregulation (A) and CD69 upregulation (B) by Jurkat 76 cells transduced with mo5 Vγ7Vδ2-2 TCR and co-cultured for 5 h with MODE-K.FLAG-l1.HA-l6 cells in the presence of the indicated concentrations of antibodies (x axis). Results were normalized to those obtained by co-culture with transduced MODE-K.EV cells. Data are representative of three independent experiments (mean ± SD of n = 3 co-cultures). (C) Specific staining of anti-His antibody alone (top row), soluble Vγ7 + TCR and anti-His mAb (middle row), or Vγ4 + TCR and anti-His mAb (bottom row) to 293T cells expressing Btnl1.6, BTNL3.8, or control 293T.EV. (D) Flow cytometry analysis of the staining of Btnl1.6-expressing 293T cells with increasing concentrations of soluble Vγ7 + TCR and anti-His mAb. See also Figure S2 .

    Journal: Immunity

    Article Title: Butyrophilin-like 3 Directly Binds a Human Vγ4+ T Cell Receptor Using a Modality Distinct from Clonally-Restricted Antigen

    doi: 10.1016/j.immuni.2019.09.006

    Figure Lengend Snippet: Mouse Vγ7 TCR-Dependent Recognition of Btnl1.6 (A and B) Flow cytometry analysis of TCR downregulation (A) and CD69 upregulation (B) by Jurkat 76 cells transduced with mo5 Vγ7Vδ2-2 TCR and co-cultured for 5 h with MODE-K.FLAG-l1.HA-l6 cells in the presence of the indicated concentrations of antibodies (x axis). Results were normalized to those obtained by co-culture with transduced MODE-K.EV cells. Data are representative of three independent experiments (mean ± SD of n = 3 co-cultures). (C) Specific staining of anti-His antibody alone (top row), soluble Vγ7 + TCR and anti-His mAb (middle row), or Vγ4 + TCR and anti-His mAb (bottom row) to 293T cells expressing Btnl1.6, BTNL3.8, or control 293T.EV. (D) Flow cytometry analysis of the staining of Btnl1.6-expressing 293T cells with increasing concentrations of soluble Vγ7 + TCR and anti-His mAb. See also Figure S2 .

    Article Snippet: Flow Cytometry Flow cytometry was performed using the following antibodies from BioLegend, unless otherwise stated.

    Techniques: Flow Cytometry, Cytometry, Transduction, Cell Culture, Co-Culture Assay, Staining, Expressing

    CD36 is associated with DPP4 expression on macrophages. A B , Peritoneal macrophages were co-stained with CD36 and DPP4 and then examined by imaging flow cytometry. CD36 + and CD36 − macrophages were gated for the analysis of DPP4 expression. Representative images ( A ) and histograms ( B ) showing the expression of DPP4 on CD36 + and CD36 − macrophages. C , Human MDMs were co-stained with CD36 and DPP4 and DPP4 expression on CD36 + or CD36 − MDMs are shown.

    Journal: EBioMedicine

    Article Title: Oxidized LDL upregulates macrophage DPP4 expression via TLR4/TRIF/CD36 pathways

    doi: 10.1016/j.ebiom.2019.01.065

    Figure Lengend Snippet: CD36 is associated with DPP4 expression on macrophages. A B , Peritoneal macrophages were co-stained with CD36 and DPP4 and then examined by imaging flow cytometry. CD36 + and CD36 − macrophages were gated for the analysis of DPP4 expression. Representative images ( A ) and histograms ( B ) showing the expression of DPP4 on CD36 + and CD36 − macrophages. C , Human MDMs were co-stained with CD36 and DPP4 and DPP4 expression on CD36 + or CD36 − MDMs are shown.

    Article Snippet: 2.6 Flow cytometry All antibodies used in imaging flow cytometry were purchased from BioLegend (San Diego, CA), BD (San Jose, CA), or R & D Systems (Minneapolis, MN).

    Techniques: Expressing, Staining, Imaging, Flow Cytometry, Cytometry

    CD36 is implicated in oxLDL-induced DPP4 up-regulation. Bone marrows isolated from wild-type (WT) or Cd36 −/− mice were used for the induction of BMMs. The expressions of DPP4 on both WT and Cd36 −/− BMMs were detected by imaging flow cytometry after 24-h treatment with 25 μg/mL oxLDL. Statistical analysis ( A ), representative images ( B ), histograms ( C ), and dot plots ( D ) are shown. *, p

    Journal: EBioMedicine

    Article Title: Oxidized LDL upregulates macrophage DPP4 expression via TLR4/TRIF/CD36 pathways

    doi: 10.1016/j.ebiom.2019.01.065

    Figure Lengend Snippet: CD36 is implicated in oxLDL-induced DPP4 up-regulation. Bone marrows isolated from wild-type (WT) or Cd36 −/− mice were used for the induction of BMMs. The expressions of DPP4 on both WT and Cd36 −/− BMMs were detected by imaging flow cytometry after 24-h treatment with 25 μg/mL oxLDL. Statistical analysis ( A ), representative images ( B ), histograms ( C ), and dot plots ( D ) are shown. *, p

    Article Snippet: 2.6 Flow cytometry All antibodies used in imaging flow cytometry were purchased from BioLegend (San Diego, CA), BD (San Jose, CA), or R & D Systems (Minneapolis, MN).

    Techniques: Isolation, Mouse Assay, Imaging, Flow Cytometry, Cytometry

    Oxidized low-density lipoprotein (oxLDL) increased DPP4 expression. A , Bone marrow cells isolated from C57BL/6 mice were cultured in the presence of 10 ng/mL M-CSF for 5 days and treated with 25 μg/mL native LDL or oxLDL for 24 h. Cells were then collected for the flow cytometric detection of DPP4 expression, DPP4 expression on bone marrow derived macrophages (BMMs) was detected by flow cytometry. Representative dot plots and statistical bar graph are shown. B , The DPP4 + macrophages were next gated for the analysis of DPP4 MFI. Histograms and bar graph show the DPP4 MFI on DPP4 + macrophages under different treatment conditions. C , Peritoneal macrophages were isolated from C57BL/6 mice and treated with 25 μg/mL oxLDL or vehicle for 24 h. Representative histograms show the expression of DPP4 on macrophages. D , Bone marrow-derived macrophages from C57BL/6 mice were treated with 25 μg/mL oxLDL for 24 h and the expression inflammatory genes was examined using quantitative real-time PCR. E , Monocytes were isolated from human peripheral blood and cultured in the presence of 10 ng/mL M-CSF for 5 days. Cells were then treated with 25 μg/mL oxLDL or vehicle for 24 h. Representative dot plots and histograms are shown. *, p

    Journal: EBioMedicine

    Article Title: Oxidized LDL upregulates macrophage DPP4 expression via TLR4/TRIF/CD36 pathways

    doi: 10.1016/j.ebiom.2019.01.065

    Figure Lengend Snippet: Oxidized low-density lipoprotein (oxLDL) increased DPP4 expression. A , Bone marrow cells isolated from C57BL/6 mice were cultured in the presence of 10 ng/mL M-CSF for 5 days and treated with 25 μg/mL native LDL or oxLDL for 24 h. Cells were then collected for the flow cytometric detection of DPP4 expression, DPP4 expression on bone marrow derived macrophages (BMMs) was detected by flow cytometry. Representative dot plots and statistical bar graph are shown. B , The DPP4 + macrophages were next gated for the analysis of DPP4 MFI. Histograms and bar graph show the DPP4 MFI on DPP4 + macrophages under different treatment conditions. C , Peritoneal macrophages were isolated from C57BL/6 mice and treated with 25 μg/mL oxLDL or vehicle for 24 h. Representative histograms show the expression of DPP4 on macrophages. D , Bone marrow-derived macrophages from C57BL/6 mice were treated with 25 μg/mL oxLDL for 24 h and the expression inflammatory genes was examined using quantitative real-time PCR. E , Monocytes were isolated from human peripheral blood and cultured in the presence of 10 ng/mL M-CSF for 5 days. Cells were then treated with 25 μg/mL oxLDL or vehicle for 24 h. Representative dot plots and histograms are shown. *, p

    Article Snippet: 2.6 Flow cytometry All antibodies used in imaging flow cytometry were purchased from BioLegend (San Diego, CA), BD (San Jose, CA), or R & D Systems (Minneapolis, MN).

    Techniques: Expressing, Isolation, Mouse Assay, Cell Culture, Flow Cytometry, Derivative Assay, Cytometry, Real-time Polymerase Chain Reaction

    MyD88 signaling is not responsible for oxLDL-induced DPP4 up-regulation. Bone marrows isolated from wild-type (WT) or Myd88 −/− mice were used for the induction of BMMs. The expressions of DPP4 on both WT and Myd88 −/− BMMs were detected by imaging flow cytometry after 24-h treatment of 25 μg/mL oxLDL. Representative images ( A ), histograms ( B ) and statistical analysis of DPP4 + macrophage frequency ( C ) or DPP4 MFI ( D ) are shown. *, p

    Journal: EBioMedicine

    Article Title: Oxidized LDL upregulates macrophage DPP4 expression via TLR4/TRIF/CD36 pathways

    doi: 10.1016/j.ebiom.2019.01.065

    Figure Lengend Snippet: MyD88 signaling is not responsible for oxLDL-induced DPP4 up-regulation. Bone marrows isolated from wild-type (WT) or Myd88 −/− mice were used for the induction of BMMs. The expressions of DPP4 on both WT and Myd88 −/− BMMs were detected by imaging flow cytometry after 24-h treatment of 25 μg/mL oxLDL. Representative images ( A ), histograms ( B ) and statistical analysis of DPP4 + macrophage frequency ( C ) or DPP4 MFI ( D ) are shown. *, p

    Article Snippet: 2.6 Flow cytometry All antibodies used in imaging flow cytometry were purchased from BioLegend (San Diego, CA), BD (San Jose, CA), or R & D Systems (Minneapolis, MN).

    Techniques: Isolation, Mouse Assay, Imaging, Flow Cytometry, Cytometry

    Inhibition of miR-155 in EMPs influences T lymphocytes differentiation in aGVHD mice After being subjected to lethal total body irradiation, BALB/c mice were transplanted intravenously with 1 × 10 7 BM cells and 2 × 10 7 spleen cells isolated from C57BL/6 donors to establish aGVHD model. EMPs (60 μg) from mouse TNF-α-stimulated MAECs, TNF-α-stimulated MAECs protected by 2.5 μmol/L simvastatin, TNF-α-stimulated MAECs transfected by antagomir-155, TNF-α-stimulated MAECs transfected by antagomir-NC were given intravenously to aGVHD mice on day0 and +7d, retrospectively. Antagomir-155 and antagomir-NC of 25 mg/kg was given intravenously on +7d followed by 5 mg/kg twice weekly up to +21d. Peripheral blood T lymphocytes subsets were assessed by flow cytometry analysis on +21d. Shown is the mean ± SD from three combined independent experiments. * P

    Journal: Oncotarget

    Article Title: Endothelial microparticles delivering microRNA-155 into T lymphocytes are involved in the initiation of acute graft-versus-host disease following allogeneic hematopoietic stem cell transplantation

    doi: 10.18632/oncotarget.15579

    Figure Lengend Snippet: Inhibition of miR-155 in EMPs influences T lymphocytes differentiation in aGVHD mice After being subjected to lethal total body irradiation, BALB/c mice were transplanted intravenously with 1 × 10 7 BM cells and 2 × 10 7 spleen cells isolated from C57BL/6 donors to establish aGVHD model. EMPs (60 μg) from mouse TNF-α-stimulated MAECs, TNF-α-stimulated MAECs protected by 2.5 μmol/L simvastatin, TNF-α-stimulated MAECs transfected by antagomir-155, TNF-α-stimulated MAECs transfected by antagomir-NC were given intravenously to aGVHD mice on day0 and +7d, retrospectively. Antagomir-155 and antagomir-NC of 25 mg/kg was given intravenously on +7d followed by 5 mg/kg twice weekly up to +21d. Peripheral blood T lymphocytes subsets were assessed by flow cytometry analysis on +21d. Shown is the mean ± SD from three combined independent experiments. * P

    Article Snippet: T lymphocytes subset analysis The following flow cytometry antibodies were used to stain the T lymphocytes subsets, including anti-CD4-FITC, anti-CD25-PerCP-Cy5.5, anti-FOXP3-Alexa-Fluor647, and anti-IL-17A-PE for human T cells and anti-CD3-APC, anti-CD4-PE/Cy7, anti-CD8-FITC, anti-CD25-APC, anti-FOXP3-PE, and anti-IL-17A-PE for mouse T cells (BioLegend, San Diego, USA).

    Techniques: Inhibition, Mouse Assay, Irradiation, Isolation, Transfection, Flow Cytometry, Cytometry

    miR-155 in EMPs promotes Th1, Th9 and Th17 and inhibit Th2 and Treg cells differentiation Purified T lymphocytes were co-cultured with 10 μg/mL EMPs from EA.hy926, TNF-α-stimulated EA.hy926, TNF-α-stimulated EA.hy926 protected by 2.5 μmol/L simvastatin, TNF-α-stimulated EA.hy926 transinfected by antagomir-155 and TNF-α-stimulated EA.hy926 transinfected by antagomir-NC, respectively. Cytokines were determined using ELISA kits 3 days after co-culture. T lymphocytes subsets were analyzed by flow cytometry. Data represent mean ± SD from three independent experiments. * P

    Journal: Oncotarget

    Article Title: Endothelial microparticles delivering microRNA-155 into T lymphocytes are involved in the initiation of acute graft-versus-host disease following allogeneic hematopoietic stem cell transplantation

    doi: 10.18632/oncotarget.15579

    Figure Lengend Snippet: miR-155 in EMPs promotes Th1, Th9 and Th17 and inhibit Th2 and Treg cells differentiation Purified T lymphocytes were co-cultured with 10 μg/mL EMPs from EA.hy926, TNF-α-stimulated EA.hy926, TNF-α-stimulated EA.hy926 protected by 2.5 μmol/L simvastatin, TNF-α-stimulated EA.hy926 transinfected by antagomir-155 and TNF-α-stimulated EA.hy926 transinfected by antagomir-NC, respectively. Cytokines were determined using ELISA kits 3 days after co-culture. T lymphocytes subsets were analyzed by flow cytometry. Data represent mean ± SD from three independent experiments. * P

    Article Snippet: T lymphocytes subset analysis The following flow cytometry antibodies were used to stain the T lymphocytes subsets, including anti-CD4-FITC, anti-CD25-PerCP-Cy5.5, anti-FOXP3-Alexa-Fluor647, and anti-IL-17A-PE for human T cells and anti-CD3-APC, anti-CD4-PE/Cy7, anti-CD8-FITC, anti-CD25-APC, anti-FOXP3-PE, and anti-IL-17A-PE for mouse T cells (BioLegend, San Diego, USA).

    Techniques: Purification, Cell Culture, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Flow Cytometry, Cytometry

    miR-155 expressions in MPs, plasma and T lymphocytes of peripheral blood from BM and aGVHD mice before allo-BMT, at +4d, +8d, +12d, +16d and aGVHD point Average values ± SD from three independent experiments were plotted. ( A ) EMPs isolated from the peripheral blood of BM and aGVHD groups were assessed by flow cytometry analysis. * P

    Journal: Oncotarget

    Article Title: Endothelial microparticles delivering microRNA-155 into T lymphocytes are involved in the initiation of acute graft-versus-host disease following allogeneic hematopoietic stem cell transplantation

    doi: 10.18632/oncotarget.15579

    Figure Lengend Snippet: miR-155 expressions in MPs, plasma and T lymphocytes of peripheral blood from BM and aGVHD mice before allo-BMT, at +4d, +8d, +12d, +16d and aGVHD point Average values ± SD from three independent experiments were plotted. ( A ) EMPs isolated from the peripheral blood of BM and aGVHD groups were assessed by flow cytometry analysis. * P

    Article Snippet: T lymphocytes subset analysis The following flow cytometry antibodies were used to stain the T lymphocytes subsets, including anti-CD4-FITC, anti-CD25-PerCP-Cy5.5, anti-FOXP3-Alexa-Fluor647, and anti-IL-17A-PE for human T cells and anti-CD3-APC, anti-CD4-PE/Cy7, anti-CD8-FITC, anti-CD25-APC, anti-FOXP3-PE, and anti-IL-17A-PE for mouse T cells (BioLegend, San Diego, USA).

    Techniques: Mouse Assay, Isolation, Flow Cytometry, Cytometry

    VHH16 and VHH21 recognize mouse Ly-6C and Ly-6G. ( A – D ) Mouse BMDC were obtained after differentiation for 6 days with GM-CSF and IL-4. ( A ) MHCII vs CD11c expression on live, differentiated cells. ( B ) Binding of GFP-specific Enhancer, VHH16 and VHH21 on live cells. ( C ) MHCII vs CD11c expression on live cells negative or positive for VHH binding. ( D ) VHH binding vs Ly-6C expression on live cells. ( E ) Ly-6C, VHH16 or VHH21 vs Ly-6G expression gated on CD11b positive fresh bone cells. ( F ) Binding of Enhancer, VHH16 or VHH21 to control HEK 293 cells or cells transfected with Ly6c1 , Ly6c2 or Ly6g constructs. Light grey histograms: unstained control. ( G and H ) Scattered plots show VHHs mean fluorescent intensity (MFI) binding on flow cytometry-sorted monocytic (CD11b + Ly-6C + Ly-6G − ) ( G ) or granulocytes (CD11b + Ly-6C low Ly-6G + ) ( H ) subsets from fresh bone marrow for indicated VHH concentrations. Each panel representative of 2 independent experiments or more.

    Journal: Scientific Reports

    Article Title: The activity of myeloid cell-specific VHH immunotoxins is target-, epitope-, subset- and organ dependent

    doi: 10.1038/s41598-017-17948-0

    Figure Lengend Snippet: VHH16 and VHH21 recognize mouse Ly-6C and Ly-6G. ( A – D ) Mouse BMDC were obtained after differentiation for 6 days with GM-CSF and IL-4. ( A ) MHCII vs CD11c expression on live, differentiated cells. ( B ) Binding of GFP-specific Enhancer, VHH16 and VHH21 on live cells. ( C ) MHCII vs CD11c expression on live cells negative or positive for VHH binding. ( D ) VHH binding vs Ly-6C expression on live cells. ( E ) Ly-6C, VHH16 or VHH21 vs Ly-6G expression gated on CD11b positive fresh bone cells. ( F ) Binding of Enhancer, VHH16 or VHH21 to control HEK 293 cells or cells transfected with Ly6c1 , Ly6c2 or Ly6g constructs. Light grey histograms: unstained control. ( G and H ) Scattered plots show VHHs mean fluorescent intensity (MFI) binding on flow cytometry-sorted monocytic (CD11b + Ly-6C + Ly-6G − ) ( G ) or granulocytes (CD11b + Ly-6C low Ly-6G + ) ( H ) subsets from fresh bone marrow for indicated VHH concentrations. Each panel representative of 2 independent experiments or more.

    Article Snippet: Flow Cytometry Antibody staining was done in presence of Fc receptor blockade (TruStain fcX BioLegend, #101320) in FACS buffer (PBS with 1% FBS).

    Techniques: Expressing, Binding Assay, Transfection, Construct, Flow Cytometry, Cytometry

    ALX148 binds human CD47 and prevents its interaction with SIRPα. (A) Structure of ALX148 molecule. Lines indicate disulfide bonds between ALX148 monomers. (B) Flow cytometry analysis of ALX148 binding (solid white histograms), negative control protein ALX180 binding (dashed histograms), or background fluorescence of unstained cells (filled histograms) for indicated human and mouse tumor cell lines. Cell number is normalized to mode. Results are representative of at least three independent experiments. (C) Flow cytometry analysis of wild-type SIRPα binding to Jurkat cells. Geometric mean fluorescence intensity is indicated on the y-axis. Cells were pre-incubated with proteins indicated on the x-axis prior to SIRPα incubation. Bkgd = background fluorescence in the absence of labeled wild-type SIRPα. Error bars indicate standard deviation of triplicates. Results are representative of three independent experiments.

    Journal: PLoS ONE

    Article Title: ALX148 blocks CD47 and enhances innate and adaptive antitumor immunity with a favorable safety profile

    doi: 10.1371/journal.pone.0201832

    Figure Lengend Snippet: ALX148 binds human CD47 and prevents its interaction with SIRPα. (A) Structure of ALX148 molecule. Lines indicate disulfide bonds between ALX148 monomers. (B) Flow cytometry analysis of ALX148 binding (solid white histograms), negative control protein ALX180 binding (dashed histograms), or background fluorescence of unstained cells (filled histograms) for indicated human and mouse tumor cell lines. Cell number is normalized to mode. Results are representative of at least three independent experiments. (C) Flow cytometry analysis of wild-type SIRPα binding to Jurkat cells. Geometric mean fluorescence intensity is indicated on the y-axis. Cells were pre-incubated with proteins indicated on the x-axis prior to SIRPα incubation. Bkgd = background fluorescence in the absence of labeled wild-type SIRPα. Error bars indicate standard deviation of triplicates. Results are representative of three independent experiments.

    Article Snippet: Flow cytometry antibodies Antibodies used to characterize cell surface and intracellular proteins were purchased from Biolegend, BD Biosciences or eBioscience.

    Techniques: Flow Cytometry, Cytometry, Binding Assay, Negative Control, Fluorescence, Incubation, Labeling, Standard Deviation

    Characterization of MHC-II(+) melanoma cell lines. Melanoma cell lines were treated with IFNγ for 24 h before collection and live-cell staining and flow cytometry analysis for MHC-I/HLA-A/B/C ( a ), MHC-II/HLA-DR ( b ) and PD-L1 ( c ). Bars represent mean±s.e.m. for at least three experiments ( d ) Representative flow plots from c . ( e ) Western blot analysis of melanoma cell lines after 24 or 48 h of IFNγ stimulation. ( f ) Phosphorylation of STAT1 (top row) and STAT5 (bottom row) in melanoma cell lines at 15 min after IFNγ stimulation. Histograms were coloured according to the arcsinh transformed ratio or MFI medians relative to the table minimum value.

    Journal: Nature Communications

    Article Title: Melanoma-specific MHC-II expression represents a tumour-autonomous phenotype and predicts response to anti-PD-1/PD-L1 therapy

    doi: 10.1038/ncomms10582

    Figure Lengend Snippet: Characterization of MHC-II(+) melanoma cell lines. Melanoma cell lines were treated with IFNγ for 24 h before collection and live-cell staining and flow cytometry analysis for MHC-I/HLA-A/B/C ( a ), MHC-II/HLA-DR ( b ) and PD-L1 ( c ). Bars represent mean±s.e.m. for at least three experiments ( d ) Representative flow plots from c . ( e ) Western blot analysis of melanoma cell lines after 24 or 48 h of IFNγ stimulation. ( f ) Phosphorylation of STAT1 (top row) and STAT5 (bottom row) in melanoma cell lines at 15 min after IFNγ stimulation. Histograms were coloured according to the arcsinh transformed ratio or MFI medians relative to the table minimum value.

    Article Snippet: Standard flow cytometry Flow cytometry was performed using the following antibodies: HLA-DR/PE-Cy7 (Biolegend, clone L243, 1:20), CD274/PD-L1/APC (Biolegend, clone 29E.2A3, 1:200) and HLA-A/B/C –Alexa Fluor488 (1:100, Biolegend, clone W6/32) mouse MHC-II (I-A/I-E, 1:20, Biolegend, clone M5/114.15.2).

    Techniques: Staining, Flow Cytometry, Cytometry, Western Blot, Transformation Assay

    The level of IRF4 phosphorylation correlates with LMP1 and is dampened by the Src-specific inhibitor PP2 in EBV + cells. ( a ) Cells were collected, washed and fixed after treatment with 15 μM PP2 or DMSO (control) for 24 h. Cells were then permeabilized, and incubated with mouse anti-LMP1 antibody (Dako) and rabbit anti-p-IRF4 antibody (21st Century Biochemicals). After wash, cells were incubated with anti-mouse IgG APC (eBioscience) and anti-rabbit PE (eBioscience) before subjected to flow cytometry analysis. Q4 represents the portion of LMP1 + /p-IRF4 + cells. ( b ) SavIII cells were transfected with p85 shRNA (#1) (or control). shRNA expression was induced by 1 μg/ml doxycycline for 3 days. p-IRF4 was then evaluated by flow. P = 0.0007 (unpaired t -test). ( c ) BJAB cells were transfected with IRF4, IRF4 plus LMP1 (or c-Src as a positive control). Cells were subjected to flow analysis after 48 h. Statistical analysis was performed for three independent flow analyses. Results are the averages ± s.d. Representative results from at least three independent experiments are shown.

    Journal: Oncogene

    Article Title: LMP1 signaling pathway activates IRF4 in latent EBV infection and a positive circuit between PI3K and Src is required

    doi: 10.1038/onc.2016.380

    Figure Lengend Snippet: The level of IRF4 phosphorylation correlates with LMP1 and is dampened by the Src-specific inhibitor PP2 in EBV + cells. ( a ) Cells were collected, washed and fixed after treatment with 15 μM PP2 or DMSO (control) for 24 h. Cells were then permeabilized, and incubated with mouse anti-LMP1 antibody (Dako) and rabbit anti-p-IRF4 antibody (21st Century Biochemicals). After wash, cells were incubated with anti-mouse IgG APC (eBioscience) and anti-rabbit PE (eBioscience) before subjected to flow cytometry analysis. Q4 represents the portion of LMP1 + /p-IRF4 + cells. ( b ) SavIII cells were transfected with p85 shRNA (#1) (or control). shRNA expression was induced by 1 μg/ml doxycycline for 3 days. p-IRF4 was then evaluated by flow. P = 0.0007 (unpaired t -test). ( c ) BJAB cells were transfected with IRF4, IRF4 plus LMP1 (or c-Src as a positive control). Cells were subjected to flow analysis after 48 h. Statistical analysis was performed for three independent flow analyses. Results are the averages ± s.d. Representative results from at least three independent experiments are shown.

    Article Snippet: Cell pellets were washed with phosphate-buffered saline and fixed in fixation buffer (Biolegend, San Diego, CA, USA), permeabilized with permeabilization solution (eBioscience), and then incubated with mouse LMP1 CS1-4 and rabbit p-IRF4(Y121/124) antibodies for 40 min. After washing with flow cytometry buffer, cells were incubated further with anti-mouse IgG APC and anti-rabbit IgG PE for 25 min, and then followed by flow cytometry analysis with a Flow Cytometer (Accuri, BD Biosciences, Franklin Lakes, NJ, USA) and BD Accuri C6 Software (BD Biosciences, San Jose, CA, USA).

    Techniques: Incubation, Flow Cytometry, Cytometry, Transfection, shRNA, Expressing, Positive Control

    Flow cytometry analysis of cell surface marker expression. (A) Expression of surface markers CD14, CD19, CD34, CD45, HLA-DR, CD73, CD90, CD105 (filled) with relevant isotype control (unfilled). (B) Quantification of the percentage of positive events compared to the relevant isotype control. Markers set to exclude 99% of isotype control events; ≥ 4x10 4 events were collected per sample. OK3 was tested at PD10, OK3H at PD50, BMA13 at PD6, BMA13H at PD18, 1C6 at PD44 and 1C6H at PD118.

    Journal: PLoS ONE

    Article Title: Immortalisation with hTERT Impacts on Sulphated Glycosaminoglycan Secretion and Immunophenotype in a Variable and Cell Specific Manner

    doi: 10.1371/journal.pone.0133745

    Figure Lengend Snippet: Flow cytometry analysis of cell surface marker expression. (A) Expression of surface markers CD14, CD19, CD34, CD45, HLA-DR, CD73, CD90, CD105 (filled) with relevant isotype control (unfilled). (B) Quantification of the percentage of positive events compared to the relevant isotype control. Markers set to exclude 99% of isotype control events; ≥ 4x10 4 events were collected per sample. OK3 was tested at PD10, OK3H at PD50, BMA13 at PD6, BMA13H at PD18, 1C6 at PD44 and 1C6H at PD118.

    Article Snippet: Flow cytometry immunophenotyping Cells were trypsinised, counted and re-suspended in flow cytometry buffer (R & D systems) for 15 minutes and approximately 1X105 cells aliquoted into 1.5 mL microcentrifuge tubes, centrifuged at 300 g for 10 minutes and the supernatant discarded.

    Techniques: Flow Cytometry, Cytometry, Marker, Expressing

    Characterization of gp130-dNG expression and function in stably transduced BAF3 cell pools. A , flow cytometry analysis of total gp130-EYFP and gp130-dNG-EYFP expression (fluorescein isothiocyanate/GFP channel; black ) versus the nonspecific signal of untransduced

    Journal:

    Article Title: N-Linked Glycosylation Is Essential for the Stability but Not the Signaling Function of the Interleukin-6 Signal Transducer Glycoprotein 130 *

    doi: 10.1074/jbc.M109.075952

    Figure Lengend Snippet: Characterization of gp130-dNG expression and function in stably transduced BAF3 cell pools. A , flow cytometry analysis of total gp130-EYFP and gp130-dNG-EYFP expression (fluorescein isothiocyanate/GFP channel; black ) versus the nonspecific signal of untransduced

    Article Snippet: 5 × 105 stably transduced BAF3 cells were washed once (centrifugation for 5 min, ∼500 × g , 4 °C) in flow cytometry buffer (PBS containing 1% BSA (PAA Laboratories) and 0.01% sodium azide (Sigma)) and were used either directly for flow cytometry analysis of the EYFP signal in the fluorescein isothiocyanate/GFP channel or were stained with antibodies raised against human gp130 (clone B-R3 from Abcam (Cambridge, UK) or clone B-P4 from Diaclone), followed by anti-mouse IgG-allophycocyanin (BD Biosciences), according to standard protocols of the manufacturers.

    Techniques: Expressing, Stable Transfection, Flow Cytometry, Cytometry

    Lung cells infected by CAL- and HA mut viruses. Five Balb/c female mice/condition of 6–9 weeks of age were infected with 10 3 pfu of CAL and HA-mut viruses or mock infected and pooled into one sample. At days 1, 2, and 3 post-infection lungs were collected, and processed for flow cytometry. Single cell suspension was stained with antibodies against NP, EpCam and CD45, to detect infected cells, epithelial cells and leukocytes, respectively. (A) Shows events positive for NP in the y-axis. (B,C) Percentage of specific populations of infected cells in relation to the total events, total number of gated EpCam or of CD45 positive cells were plotted. (D) Total percentage of CD45 positive cells, regardless of the infectivity state were plotted. The experiment was performed twice and overall 5 mice per condition pooled into one sample. (E,F) Mice ( n = 5) were inoculated intranasally with a sublethal dose (10 3 pfu) of each recombinant virus or PBS (MOCK) as control and were evaluated in three groups. Neutrophils (E) and alveolar macrophages (F) were quantified in the lungs (% of cells) at indicated dpi. Significance was determined by Student's t -test ( * p

    Journal: Frontiers in Immunology

    Article Title: Mutation S110L of H1N1 Influenza Virus Hemagglutinin: A Potent Determinant of Attenuation in the Mouse Model

    doi: 10.3389/fimmu.2019.00132

    Figure Lengend Snippet: Lung cells infected by CAL- and HA mut viruses. Five Balb/c female mice/condition of 6–9 weeks of age were infected with 10 3 pfu of CAL and HA-mut viruses or mock infected and pooled into one sample. At days 1, 2, and 3 post-infection lungs were collected, and processed for flow cytometry. Single cell suspension was stained with antibodies against NP, EpCam and CD45, to detect infected cells, epithelial cells and leukocytes, respectively. (A) Shows events positive for NP in the y-axis. (B,C) Percentage of specific populations of infected cells in relation to the total events, total number of gated EpCam or of CD45 positive cells were plotted. (D) Total percentage of CD45 positive cells, regardless of the infectivity state were plotted. The experiment was performed twice and overall 5 mice per condition pooled into one sample. (E,F) Mice ( n = 5) were inoculated intranasally with a sublethal dose (10 3 pfu) of each recombinant virus or PBS (MOCK) as control and were evaluated in three groups. Neutrophils (E) and alveolar macrophages (F) were quantified in the lungs (% of cells) at indicated dpi. Significance was determined by Student's t -test ( * p

    Article Snippet: After this incubation, the lung cell suspension was centrifuged and then resuspended in flow cytometry buffer [PBS supplemented with 2% FBS (Life Technologies, 10500-064)].

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

    Silencing of Gal3 shifts CSC subset to a Gal3 negative CSC subset. ( a ) Western blot analysis for comparison of total Gal3 in LiM6TR or DLD1TR colon cancer cells and L3.6pl or AsPC1 pancreatic cancer cells after infection with lentiviral particles for control-shRNA ( c ) or Gal3-shRNA (G). Each pair of control and Gal3 knockdown cells were run side by side on the same gel. Comparison of Gal3 expression between cell lines has been previously published 10 ( b ) Flow cytometric analysis of colon cancer and pancreatic cancer cells after infection with lentiviral particles for control-shRNA (red trace) or Gal3-shRNA (green trace). Black trace is background staining. ( c ) ALDH activity was analyzed by flow cytometry. ALDEFLUOR activity in colon cancer and pancreatic cancer cells after infection with lentiviral particles for control-shRNA (blue trace) or Gal3-shRNA (green trace). Gray profile is background staining

    Journal: Cell Death & Disease

    Article Title: Cell surface galectin-3 defines a subset of chemoresistant gastrointestinal tumor-initiating cancer cells with heightened stem cell characteristics

    doi: 10.1038/cddis.2016.239

    Figure Lengend Snippet: Silencing of Gal3 shifts CSC subset to a Gal3 negative CSC subset. ( a ) Western blot analysis for comparison of total Gal3 in LiM6TR or DLD1TR colon cancer cells and L3.6pl or AsPC1 pancreatic cancer cells after infection with lentiviral particles for control-shRNA ( c ) or Gal3-shRNA (G). Each pair of control and Gal3 knockdown cells were run side by side on the same gel. Comparison of Gal3 expression between cell lines has been previously published 10 ( b ) Flow cytometric analysis of colon cancer and pancreatic cancer cells after infection with lentiviral particles for control-shRNA (red trace) or Gal3-shRNA (green trace). Black trace is background staining. ( c ) ALDH activity was analyzed by flow cytometry. ALDEFLUOR activity in colon cancer and pancreatic cancer cells after infection with lentiviral particles for control-shRNA (blue trace) or Gal3-shRNA (green trace). Gray profile is background staining

    Article Snippet: Flow cytometry analysis and sorting To identify and isolate CSC, parental cells were collected in Versene solution (Invitrogen, Carlsbad, CA, USA, cat#15040), washed and blocked with flow cytometry buffer (Santa Cruz Biotechnology, Dallas, TX, USA), labeled with the following primary conjugated antibodies and their matched isotypes: anti-CD24-PE-Cy7(561646), anti-EpCAM-FITC (BD Bioscience, San Jose, CA, USA, 347197) anti-CD44-APC-eFluor 780(47044182), anti-CD166-PerCPeFluor 710(46166842), anti Gal3-PE(12-5301-83) (all from - eBioscience, San Diego, CA, USA).

    Techniques: Western Blot, Infection, shRNA, Expressing, Flow Cytometry, Staining, Activity Assay, Cytometry

    Surface Gal3 defines a subtype of epithelial CSC. ( a ) Colorectal cancer cells LiM6 and DLD1 were investigated by flow cytometry for cell surface markers CD24/CD44 (left). CD24+/CD44+-cells (in black) were then investigated for CD166/EpCAM-expression (middle) and CD24+/CD44+/CD166+/EpCAM+-positive cells (in black middle) for Gal3-expression (right panel), where ++++ Gal3 − in green refers to cells positive for CD24, CD44, CD166, and EpCAM, but negative for Gal3, and ++++ Gal3 + in red refers to cells positive for CD24, CD44, CD166, EpCAM, and Gal3. ( b ) Pancreatic cancer cell lines AsPC1 and L3.6pl were subjected to the same analysis as in ( a )

    Journal: Cell Death & Disease

    Article Title: Cell surface galectin-3 defines a subset of chemoresistant gastrointestinal tumor-initiating cancer cells with heightened stem cell characteristics

    doi: 10.1038/cddis.2016.239

    Figure Lengend Snippet: Surface Gal3 defines a subtype of epithelial CSC. ( a ) Colorectal cancer cells LiM6 and DLD1 were investigated by flow cytometry for cell surface markers CD24/CD44 (left). CD24+/CD44+-cells (in black) were then investigated for CD166/EpCAM-expression (middle) and CD24+/CD44+/CD166+/EpCAM+-positive cells (in black middle) for Gal3-expression (right panel), where ++++ Gal3 − in green refers to cells positive for CD24, CD44, CD166, and EpCAM, but negative for Gal3, and ++++ Gal3 + in red refers to cells positive for CD24, CD44, CD166, EpCAM, and Gal3. ( b ) Pancreatic cancer cell lines AsPC1 and L3.6pl were subjected to the same analysis as in ( a )

    Article Snippet: Flow cytometry analysis and sorting To identify and isolate CSC, parental cells were collected in Versene solution (Invitrogen, Carlsbad, CA, USA, cat#15040), washed and blocked with flow cytometry buffer (Santa Cruz Biotechnology, Dallas, TX, USA), labeled with the following primary conjugated antibodies and their matched isotypes: anti-CD24-PE-Cy7(561646), anti-EpCAM-FITC (BD Bioscience, San Jose, CA, USA, 347197) anti-CD44-APC-eFluor 780(47044182), anti-CD166-PerCPeFluor 710(46166842), anti Gal3-PE(12-5301-83) (all from - eBioscience, San Diego, CA, USA).

    Techniques: Flow Cytometry, Cytometry, Expressing

    Estrogen abrogates HCV infection. (A) Huh7.5 cells respond to estrogen treatment measured by cyclin-D1 expression. (B) HCV core protein in J6/JFH1-infected Huh7.5 cells treated with E2 or Universal Type I IFN. (C) HCV intracellular core protein in J6/JFH1-infected Huh7.5 cells treated with E2, as measured by Flow Cytometry. (D) HCV RNA quantitated relative to GAPDH RNA levels from infected Huh7.5 cells treated with E2 or IFN. (E) Virus growth measured by absolute mean focus forming units (FFU). Error bars indicate the statistical standard deviation from the mean (±SD). Statistical significance is indicated by asterisks where: (*** = P ≤ 0.001; **** = P ≤ 0.0001). (F) Representative images of foci after 48 hr of E2 treatment. Magnification 40X ( below ); 100X ( above ).

    Journal: PLoS ONE

    Article Title: A New Signaling Pathway for HCV Inhibition by Estrogen: GPR30 Activation Leads to Cleavage of Occludin by MMP-9

    doi: 10.1371/journal.pone.0145212

    Figure Lengend Snippet: Estrogen abrogates HCV infection. (A) Huh7.5 cells respond to estrogen treatment measured by cyclin-D1 expression. (B) HCV core protein in J6/JFH1-infected Huh7.5 cells treated with E2 or Universal Type I IFN. (C) HCV intracellular core protein in J6/JFH1-infected Huh7.5 cells treated with E2, as measured by Flow Cytometry. (D) HCV RNA quantitated relative to GAPDH RNA levels from infected Huh7.5 cells treated with E2 or IFN. (E) Virus growth measured by absolute mean focus forming units (FFU). Error bars indicate the statistical standard deviation from the mean (±SD). Statistical significance is indicated by asterisks where: (*** = P ≤ 0.001; **** = P ≤ 0.0001). (F) Representative images of foci after 48 hr of E2 treatment. Magnification 40X ( below ); 100X ( above ).

    Article Snippet: For HCV core staining, fixation was carried out by using Flow cytometry Fixation Buffer (R & D Systems) and incubating the cells for 10 minutes at room temperature.

    Techniques: Infection, Expressing, Flow Cytometry, Cytometry, Standard Deviation

    Spore-FP1 induces enrichment of tissue resident memory cells. Mice were first immunized with Bacillus Calmette-Guérin for 6 weeks (except the PBS group) and then received two intranasal doses of either spores alone, fusion protein 1 (FP1) alone, or Spore-FP1. Lung parenchymal cells were assessed by flow cytometry for T-cell markers. A gating strategy of live cells→single cells→CD3 + →CD4 + /CD8 + →CD44 hi CD62L lo was used to measure the frequency of double-positive CD69/CD103 Trm. Data are derived from n = 3 pooled mice per group showing a representative plot.

    Journal: Frontiers in Immunology

    Article Title: Mucosal Delivery of Fusion Proteins with Bacillus subtilis Spores Enhances Protection against Tuberculosis by Bacillus Calmette-Guérin

    doi: 10.3389/fimmu.2018.00346

    Figure Lengend Snippet: Spore-FP1 induces enrichment of tissue resident memory cells. Mice were first immunized with Bacillus Calmette-Guérin for 6 weeks (except the PBS group) and then received two intranasal doses of either spores alone, fusion protein 1 (FP1) alone, or Spore-FP1. Lung parenchymal cells were assessed by flow cytometry for T-cell markers. A gating strategy of live cells→single cells→CD3 + →CD4 + /CD8 + →CD44 hi CD62L lo was used to measure the frequency of double-positive CD69/CD103 Trm. Data are derived from n = 3 pooled mice per group showing a representative plot.

    Article Snippet: For surface staining, cells were then stained in flow cytometry buffer (PBS containing 0.5% BSA and 0.1% sodium azide—all from Sigma-Aldrich) for 30–45 min at 4°C.

    Techniques: Mouse Assay, Flow Cytometry, Cytometry, Derivative Assay

    Cytokine profiles during splenocyte antigen recall. Splenocytes from immunized mice were stimulated in technical duplicates with 5 µg/mL recall antigen for 5–6 days and T-cell cytokines were measured by multiplex flow cytometry. Results are expressed as mean ± SEM. Data are derived from n = 3 pooled spleens per group.

    Journal: Frontiers in Immunology

    Article Title: Mucosal Delivery of Fusion Proteins with Bacillus subtilis Spores Enhances Protection against Tuberculosis by Bacillus Calmette-Guérin

    doi: 10.3389/fimmu.2018.00346

    Figure Lengend Snippet: Cytokine profiles during splenocyte antigen recall. Splenocytes from immunized mice were stimulated in technical duplicates with 5 µg/mL recall antigen for 5–6 days and T-cell cytokines were measured by multiplex flow cytometry. Results are expressed as mean ± SEM. Data are derived from n = 3 pooled spleens per group.

    Article Snippet: For surface staining, cells were then stained in flow cytometry buffer (PBS containing 0.5% BSA and 0.1% sodium azide—all from Sigma-Aldrich) for 30–45 min at 4°C.

    Techniques: Mouse Assay, Multiplex Assay, Flow Cytometry, Cytometry, Derivative Assay

    Bacillus subtilis spores activate antigen-presenting cells. (A) Dendritic cells (DCs) (left) and macrophages (right) were stimulated in duplicate for 48 h with LPS (100 ng/mL) or B. subtilis spores (1, 10, and 100 MOI) and surface molecule expression was measured by flow cytometry on gated viable cells. MFI was normalized to the unstimulated control. (B) Cytokines from the supernatants were tested for proinflammatory cytokine production by ELISA. (C) Macrophages were stimulated for 20 h with LPS (100 ng/mL) or B. subtilis spores (100 MOI) in the presence of brefeldin A (10 µg/mL), followed by intracellular detection of IL-12p40. EC, empty channel. A representative experiment is shown. (D) Transcription factor phosphorylation levels were determined by PhosphoFlow. Macrophages were stimulated with 100 ng/mL LPS (blue histograms) or 100 MOI spores (red histograms) for 4 h and then fixed and stained. Some cells were left untreated (black histograms). Representative MFI values are plotted on the relevant histogram. Data are from three (A–C) or one (D) independent experiments. Results are expressed as mean ± SEM. Significance was tested against the unstimulated control by one-way ANOVA with Fisher’s posttest, * p

    Journal: Frontiers in Immunology

    Article Title: Mucosal Delivery of Fusion Proteins with Bacillus subtilis Spores Enhances Protection against Tuberculosis by Bacillus Calmette-Guérin

    doi: 10.3389/fimmu.2018.00346

    Figure Lengend Snippet: Bacillus subtilis spores activate antigen-presenting cells. (A) Dendritic cells (DCs) (left) and macrophages (right) were stimulated in duplicate for 48 h with LPS (100 ng/mL) or B. subtilis spores (1, 10, and 100 MOI) and surface molecule expression was measured by flow cytometry on gated viable cells. MFI was normalized to the unstimulated control. (B) Cytokines from the supernatants were tested for proinflammatory cytokine production by ELISA. (C) Macrophages were stimulated for 20 h with LPS (100 ng/mL) or B. subtilis spores (100 MOI) in the presence of brefeldin A (10 µg/mL), followed by intracellular detection of IL-12p40. EC, empty channel. A representative experiment is shown. (D) Transcription factor phosphorylation levels were determined by PhosphoFlow. Macrophages were stimulated with 100 ng/mL LPS (blue histograms) or 100 MOI spores (red histograms) for 4 h and then fixed and stained. Some cells were left untreated (black histograms). Representative MFI values are plotted on the relevant histogram. Data are from three (A–C) or one (D) independent experiments. Results are expressed as mean ± SEM. Significance was tested against the unstimulated control by one-way ANOVA with Fisher’s posttest, * p

    Article Snippet: For surface staining, cells were then stained in flow cytometry buffer (PBS containing 0.5% BSA and 0.1% sodium azide—all from Sigma-Aldrich) for 30–45 min at 4°C.

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

    Enhanced ZIKV infection of HCs is modulated in an IgG subclass-dependent manner. A) Flow cytometry plots showing expression of FcγRIII (CD16), FcγRII (CD32), and FcγRI (CD64) of uninfected HCs. Representative experiment from n=2

    Journal: Cell host & microbe

    Article Title: Cross-reactive dengue virus antibodies augment Zika virus infection of human placental macrophages

    doi: 10.1016/j.chom.2018.10.008

    Figure Lengend Snippet: Enhanced ZIKV infection of HCs is modulated in an IgG subclass-dependent manner. A) Flow cytometry plots showing expression of FcγRIII (CD16), FcγRII (CD32), and FcγRI (CD64) of uninfected HCs. Representative experiment from n=2

    Article Snippet: HCs were fixed with 1x Transcription Factor Fix/Perm (diluted in Transcription Factor Fix/Perm Diluent; TONBO Biosciences) for 20min on ice and permeabilized by washing twice with 1x Flow Cytometry Perm Buffer (diluted in ddiH2 O; TONBO Biosciences).

    Techniques: Infection, Flow Cytometry, Cytometry, Expressing