tnfα Biolegend Search Results


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  • 97
    BioLegend tnfα
    Treatment of NP-specific CD8 + cytotoxic T lymphocyte (CTL) with EF3030-derived lysate inhibits the production of IFNγ and tumor necrosis factor α <t>(TNFα)</t> and the release of cytolytic granules. NP 147–155 -specific CTL was stimulated with peptide in the presence of increasing amounts of Spn lysate. Anti-CD107a antibody was included in the stimulation phase to identify cells releasing lytic granules. Following stimulation, cells were stained for CD8, IFNγ, and TNFα. Data shown are pregated on live CD8 + CTL. The percent inhibition refers to loss of the effector function noted in the legend. (A) Representative flow plots. (B) Averaged data ± SEM from eight independent experiments assessed using two independently generated NP-specific CTL lines.
    Tnfα, supplied by BioLegend, used in various techniques. Bioz Stars score: 97/100, based on 1003 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    BioLegend tnfα pecy7
    Treatment of NP-specific CD8 + cytotoxic T lymphocyte (CTL) with EF3030-derived lysate inhibits the production of IFNγ and tumor necrosis factor α <t>(TNFα)</t> and the release of cytolytic granules. NP 147–155 -specific CTL was stimulated with peptide in the presence of increasing amounts of Spn lysate. Anti-CD107a antibody was included in the stimulation phase to identify cells releasing lytic granules. Following stimulation, cells were stained for CD8, IFNγ, and TNFα. Data shown are pregated on live CD8 + CTL. The percent inhibition refers to loss of the effector function noted in the legend. (A) Representative flow plots. (B) Averaged data ± SEM from eight independent experiments assessed using two independently generated NP-specific CTL lines.
    Tnfα Pecy7, supplied by BioLegend, used in various techniques. Bioz Stars score: 82/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    BioLegend moab tnfα pe
    Treatment of NP-specific CD8 + cytotoxic T lymphocyte (CTL) with EF3030-derived lysate inhibits the production of IFNγ and tumor necrosis factor α <t>(TNFα)</t> and the release of cytolytic granules. NP 147–155 -specific CTL was stimulated with peptide in the presence of increasing amounts of Spn lysate. Anti-CD107a antibody was included in the stimulation phase to identify cells releasing lytic granules. Following stimulation, cells were stained for CD8, IFNγ, and TNFα. Data shown are pregated on live CD8 + CTL. The percent inhibition refers to loss of the effector function noted in the legend. (A) Representative flow plots. (B) Averaged data ± SEM from eight independent experiments assessed using two independently generated NP-specific CTL lines.
    Moab Tnfα Pe, supplied by BioLegend, used in various techniques. Bioz Stars score: 82/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    BioLegend anti tnfα
    Treatment of NP-specific CD8 + cytotoxic T lymphocyte (CTL) with EF3030-derived lysate inhibits the production of IFNγ and tumor necrosis factor α <t>(TNFα)</t> and the release of cytolytic granules. NP 147–155 -specific CTL was stimulated with peptide in the presence of increasing amounts of Spn lysate. Anti-CD107a antibody was included in the stimulation phase to identify cells releasing lytic granules. Following stimulation, cells were stained for CD8, IFNγ, and TNFα. Data shown are pregated on live CD8 + CTL. The percent inhibition refers to loss of the effector function noted in the legend. (A) Representative flow plots. (B) Averaged data ± SEM from eight independent experiments assessed using two independently generated NP-specific CTL lines.
    Anti Tnfα, supplied by BioLegend, used in various techniques. Bioz Stars score: 94/100, based on 112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    BioLegend tnfα expression
    HIF1α is critical for pro-inflammatory macrophage differentiation in vitro . Sorted PEMs from WT and HIF1α −/− mice were stimulated with LPS (100 ng/mL) or curdlan (100 ng/mL) for 10–12 h, and the indicated mRNA expression was determined with qPCR ( A ). Supernatant was collected and the concentration of the indicated cytokines was determined with an ELISA ( B ). ( C ) Peritoneal exudate cells from WT and HIF1α −/− mice were activated with the indicated stimuli, and the intracellular expression of <t>TNFα</t> in F4/80 + macrophages was determined with flow cytometry; a representative image is shown in the left figure, and data are summarized in the right figure. ( D ) Sorted PEMs were stimulated with LPS or curdlan for 10–12 h, and the mRNA expression of glycolytic pathway associated molecules was determined with qPCR. Data are presented as the means ± SD (n = 3–5). One representative experiment of three independent experiments is shown. * P
    Tnfα Expression, supplied by BioLegend, used in various techniques. Bioz Stars score: 86/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    BioLegend liver tnfα
    LPS induces miR-155 and <t>TNFα</t> expression in hepatic immune cells. Isolated LMNCs and Kupffer cells from C57Bl/6 WT mice were stimulated or not with 100ng/ml LPS for 6 hours in vitro. miR-155 expression (A: LMNCs, C: KCs) and TNF-α protein secretion (B: LMNCs, D: KCs) were measured in the cells and in the supernatant, respectively (n = 8-10/group). RNA was isolated from C57Bl/6 WT mice fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 3, 6 and 8 weeks. miR155 and TNFα mRNA expression was determined (n = 5-6/group, total 32) and correlation was plotted (E); correlation coefficient is shown. (*) indicates p
    Liver Tnfα, supplied by BioLegend, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    BioLegend tnfα elisa kits
    LPS induces miR-155 and <t>TNFα</t> expression in hepatic immune cells. Isolated LMNCs and Kupffer cells from C57Bl/6 WT mice were stimulated or not with 100ng/ml LPS for 6 hours in vitro. miR-155 expression (A: LMNCs, C: KCs) and TNF-α protein secretion (B: LMNCs, D: KCs) were measured in the cells and in the supernatant, respectively (n = 8-10/group). RNA was isolated from C57Bl/6 WT mice fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 3, 6 and 8 weeks. miR155 and TNFα mRNA expression was determined (n = 5-6/group, total 32) and correlation was plotted (E); correlation coefficient is shown. (*) indicates p
    Tnfα Elisa Kits, supplied by BioLegend, used in various techniques. Bioz Stars score: 77/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    BioLegend anti tnfα apc
    LPS induces miR-155 and <t>TNFα</t> expression in hepatic immune cells. Isolated LMNCs and Kupffer cells from C57Bl/6 WT mice were stimulated or not with 100ng/ml LPS for 6 hours in vitro. miR-155 expression (A: LMNCs, C: KCs) and TNF-α protein secretion (B: LMNCs, D: KCs) were measured in the cells and in the supernatant, respectively (n = 8-10/group). RNA was isolated from C57Bl/6 WT mice fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 3, 6 and 8 weeks. miR155 and TNFα mRNA expression was determined (n = 5-6/group, total 32) and correlation was plotted (E); correlation coefficient is shown. (*) indicates p
    Anti Tnfα Apc, supplied by BioLegend, used in various techniques. Bioz Stars score: 93/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend tnfα apc
    LPS induces miR-155 and <t>TNFα</t> expression in hepatic immune cells. Isolated LMNCs and Kupffer cells from C57Bl/6 WT mice were stimulated or not with 100ng/ml LPS for 6 hours in vitro. miR-155 expression (A: LMNCs, C: KCs) and TNF-α protein secretion (B: LMNCs, D: KCs) were measured in the cells and in the supernatant, respectively (n = 8-10/group). RNA was isolated from C57Bl/6 WT mice fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 3, 6 and 8 weeks. miR155 and TNFα mRNA expression was determined (n = 5-6/group, total 32) and correlation was plotted (E); correlation coefficient is shown. (*) indicates p
    Tnfα Apc, supplied by BioLegend, used in various techniques. Bioz Stars score: 83/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    BioLegend murine tnfα elisa kit
    LPS induces miR-155 and <t>TNFα</t> expression in hepatic immune cells. Isolated LMNCs and Kupffer cells from C57Bl/6 WT mice were stimulated or not with 100ng/ml LPS for 6 hours in vitro. miR-155 expression (A: LMNCs, C: KCs) and TNF-α protein secretion (B: LMNCs, D: KCs) were measured in the cells and in the supernatant, respectively (n = 8-10/group). RNA was isolated from C57Bl/6 WT mice fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 3, 6 and 8 weeks. miR155 and TNFα mRNA expression was determined (n = 5-6/group, total 32) and correlation was plotted (E); correlation coefficient is shown. (*) indicates p
    Murine Tnfα Elisa Kit, supplied by BioLegend, used in various techniques. Bioz Stars score: 78/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    BioLegend tnfα fitc
    LPS induces miR-155 and <t>TNFα</t> expression in hepatic immune cells. Isolated LMNCs and Kupffer cells from C57Bl/6 WT mice were stimulated or not with 100ng/ml LPS for 6 hours in vitro. miR-155 expression (A: LMNCs, C: KCs) and TNF-α protein secretion (B: LMNCs, D: KCs) were measured in the cells and in the supernatant, respectively (n = 8-10/group). RNA was isolated from C57Bl/6 WT mice fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 3, 6 and 8 weeks. miR155 and TNFα mRNA expression was determined (n = 5-6/group, total 32) and correlation was plotted (E); correlation coefficient is shown. (*) indicates p
    Tnfα Fitc, supplied by BioLegend, used in various techniques. Bioz Stars score: 79/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    BioLegend tumor necrosis factor alpha tnfα
    LPS induces miR-155 and <t>TNFα</t> expression in hepatic immune cells. Isolated LMNCs and Kupffer cells from C57Bl/6 WT mice were stimulated or not with 100ng/ml LPS for 6 hours in vitro. miR-155 expression (A: LMNCs, C: KCs) and TNF-α protein secretion (B: LMNCs, D: KCs) were measured in the cells and in the supernatant, respectively (n = 8-10/group). RNA was isolated from C57Bl/6 WT mice fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 3, 6 and 8 weeks. miR155 and TNFα mRNA expression was determined (n = 5-6/group, total 32) and correlation was plotted (E); correlation coefficient is shown. (*) indicates p
    Tumor Necrosis Factor Alpha Tnfα, supplied by BioLegend, used in various techniques. Bioz Stars score: 91/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    BioLegend anti human tnfα antibody
    LPS induces miR-155 and <t>TNFα</t> expression in hepatic immune cells. Isolated LMNCs and Kupffer cells from C57Bl/6 WT mice were stimulated or not with 100ng/ml LPS for 6 hours in vitro. miR-155 expression (A: LMNCs, C: KCs) and TNF-α protein secretion (B: LMNCs, D: KCs) were measured in the cells and in the supernatant, respectively (n = 8-10/group). RNA was isolated from C57Bl/6 WT mice fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 3, 6 and 8 weeks. miR155 and TNFα mRNA expression was determined (n = 5-6/group, total 32) and correlation was plotted (E); correlation coefficient is shown. (*) indicates p
    Anti Human Tnfα Antibody, supplied by BioLegend, used in various techniques. Bioz Stars score: 80/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    BioLegend anti mouse tnfα antibody
    LPS induces miR-155 and <t>TNFα</t> expression in hepatic immune cells. Isolated LMNCs and Kupffer cells from C57Bl/6 WT mice were stimulated or not with 100ng/ml LPS for 6 hours in vitro. miR-155 expression (A: LMNCs, C: KCs) and TNF-α protein secretion (B: LMNCs, D: KCs) were measured in the cells and in the supernatant, respectively (n = 8-10/group). RNA was isolated from C57Bl/6 WT mice fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 3, 6 and 8 weeks. miR155 and TNFα mRNA expression was determined (n = 5-6/group, total 32) and correlation was plotted (E); correlation coefficient is shown. (*) indicates p
    Anti Mouse Tnfα Antibody, supplied by BioLegend, used in various techniques. Bioz Stars score: 84/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    BioLegend anti tnfα allophycocyanin apc
    Role of neutrophils and monocytes/macrophages in DAH A , peritoneal exudate cells (PEC) and bone marrow (BM) neutrophils from wild type mice treated 14-d earlier with pristane, with or without anti-Ly6G depletion (mAb IA8), were analyzed by flow cytometry. Left , surface staining with anti-Ly6G (mAb <t>RB6-8C5)-APC-Cy7</t> and anti-CD11b-Pacific blue showing depletion of neutrophils (box) from the peritoneum but not bone marrow. Right , staining with anti-Ly6G (mAb RB6-8C5)-PE (surface stain) and <t>anti-TNFα-APC</t> (intracellular stain) showing loss of intracellular TNFα + peritoneal, but not bone marrow, Ly6G + cells. B , neutrophil depletion with anti-Ly6G antibodies. Wild type (WT) mice were treated with anti-Ly6G mAb 1-d before and 7-d after pristane treatment or left untreated (Control) (n = 6 per group). H E staining showed perivascular inflammatory cell infiltrates in controls, but not anti-Ly6G-treated mice. Efficacy of neutrophil depletion in lung was verified by staining with anti-neutrophil elastase antibodies ( right ). C , anti-F4/80 staining of macrophages in WT mice after depleting neutrophils with anti-Ly6G antibodies. F4/80 + cells are indicated by arrows. D , morphometric quantification of neutrophil elastase staining in lung from control vs. anti-Ly6G-treated mice. E , H E staining ( left ) and gross pathology ( right ) of lungs (14-d after pristane) from WT mice either treated with clodronate liposomes (CloLip, n = 8) 1-d before and 7-d after pristane or left untreated (No Rx, n = 6). Perivascular inflammatory cell infiltrates and DAH were absent after CloLip treatment. F , frequency of DAH in wild type (WT) and elastase-deficient (Ela−/−) mice 14-d after pristane treatment and in WT mice treated with anti-Ly6G antibodies (Ly6G) or with CloLip 1-d before and 7-d after pristane treatment. Pathology was assessed at 14-d. **, P
    Anti Tnfα Allophycocyanin Apc, supplied by BioLegend, used in various techniques. Bioz Stars score: 80/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    BioLegend fluorochrome conjugated antibody against tnfα
    CD8 T-cell <t>TNFα</t> secretion after in vitro stimulation with JCV VP1 peptide. Circles indicate seven healthy control subjects; triangles indicate the patient’s samples. The dotted line indicates the detection threshold. Unstim = TNFα secretion of unstimulated CD8 T-cells; stim = TNFα secretion of CD8 T-cells stimulated with JCV VP1 peptide pool for 6 h.
    Fluorochrome Conjugated Antibody Against Tnfα, supplied by BioLegend, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    BioLegend pecy7 anti mouse tnfα antibody
    CD8 T-cell <t>TNFα</t> secretion after in vitro stimulation with JCV VP1 peptide. Circles indicate seven healthy control subjects; triangles indicate the patient’s samples. The dotted line indicates the detection threshold. Unstim = TNFα secretion of unstimulated CD8 T-cells; stim = TNFα secretion of CD8 T-cells stimulated with JCV VP1 peptide pool for 6 h.
    Pecy7 Anti Mouse Tnfα Antibody, supplied by BioLegend, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    BioLegend leaf purified anti human tnfα
    CD8 T-cell <t>TNFα</t> secretion after in vitro stimulation with JCV VP1 peptide. Circles indicate seven healthy control subjects; triangles indicate the patient’s samples. The dotted line indicates the detection threshold. Unstim = TNFα secretion of unstimulated CD8 T-cells; stim = TNFα secretion of CD8 T-cells stimulated with JCV VP1 peptide pool for 6 h.
    Leaf Purified Anti Human Tnfα, supplied by BioLegend, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    BioLegend biotin anti mouse tnfα primary antibody
    Increased <t>TNFα</t> surface expression and production in ADAM10 B−/− B cells
    Biotin Anti Mouse Tnfα Primary Antibody, supplied by BioLegend, used in various techniques. Bioz Stars score: 80/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    70
    BioLegend cross reactive anti human tnfα bv650 mab11
    Increased <t>TNFα</t> surface expression and production in ADAM10 B−/− B cells
    Cross Reactive Anti Human Tnfα Bv650 Mab11, supplied by BioLegend, used in various techniques. Bioz Stars score: 70/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend anti tumor necrosis factor α tnfα blocking antibody
    Increased <t>TNFα</t> surface expression and production in ADAM10 B−/− B cells
    Anti Tumor Necrosis Factor α Tnfα Blocking Antibody, supplied by BioLegend, used in various techniques. Bioz Stars score: 83/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend α human tnfα
    Increased <t>TNFα</t> surface expression and production in ADAM10 B−/− B cells
    α Human Tnfα, supplied by BioLegend, used in various techniques. Bioz Stars score: 76/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend mouse tnfα
    Tumor necrosis factor-α gene and protein expression in the 3 × Tg mouse. (A) Fold-change (2^(− δ δCt)) is the normalized gene expression (2^(−δ Ct)) in the test sample (3 × Tg treated with Thal or 3,6′-DT) divided by the normalized gene expression (2^(−δ Ct)) in the control sample (vehicle-treated 3 × Tg). Fold-change values of less than one indicate a negative- or downregulation. Both Thal and 3,6′-DT downregulated <t>TNFα</t> gene expression but the value was significant only in the 3,6′-DT group ( P = 0.033). (B) TNFα protein levels are doubled in the cortex of 3 × Tg mice compared with Non-Tg mice. 3,6′-DT, but not Thal, reduced TNFα protein levels near to Non-Tg levels in 3 × Tg mice. n = 5 to 8 per group. One-way analysis of variance ( P = 0.062, * P
    Mouse Tnfα, supplied by BioLegend, used in various techniques. Bioz Stars score: 86/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend elisa human tnfα
    Tumor necrosis factor-α gene and protein expression in the 3 × Tg mouse. (A) Fold-change (2^(− δ δCt)) is the normalized gene expression (2^(−δ Ct)) in the test sample (3 × Tg treated with Thal or 3,6′-DT) divided by the normalized gene expression (2^(−δ Ct)) in the control sample (vehicle-treated 3 × Tg). Fold-change values of less than one indicate a negative- or downregulation. Both Thal and 3,6′-DT downregulated <t>TNFα</t> gene expression but the value was significant only in the 3,6′-DT group ( P = 0.033). (B) TNFα protein levels are doubled in the cortex of 3 × Tg mice compared with Non-Tg mice. 3,6′-DT, but not Thal, reduced TNFα protein levels near to Non-Tg levels in 3 × Tg mice. n = 5 to 8 per group. One-way analysis of variance ( P = 0.062, * P
    Elisa Human Tnfα, supplied by BioLegend, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend tnfα elisa max standard kit
    Tumor necrosis factor-α gene and protein expression in the 3 × Tg mouse. (A) Fold-change (2^(− δ δCt)) is the normalized gene expression (2^(−δ Ct)) in the test sample (3 × Tg treated with Thal or 3,6′-DT) divided by the normalized gene expression (2^(−δ Ct)) in the control sample (vehicle-treated 3 × Tg). Fold-change values of less than one indicate a negative- or downregulation. Both Thal and 3,6′-DT downregulated <t>TNFα</t> gene expression but the value was significant only in the 3,6′-DT group ( P = 0.033). (B) TNFα protein levels are doubled in the cortex of 3 × Tg mice compared with Non-Tg mice. 3,6′-DT, but not Thal, reduced TNFα protein levels near to Non-Tg levels in 3 × Tg mice. n = 5 to 8 per group. One-way analysis of variance ( P = 0.062, * P
    Tnfα Elisa Max Standard Kit, supplied by BioLegend, used in various techniques. Bioz Stars score: 78/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Treatment of NP-specific CD8 + cytotoxic T lymphocyte (CTL) with EF3030-derived lysate inhibits the production of IFNγ and tumor necrosis factor α (TNFα) and the release of cytolytic granules. NP 147–155 -specific CTL was stimulated with peptide in the presence of increasing amounts of Spn lysate. Anti-CD107a antibody was included in the stimulation phase to identify cells releasing lytic granules. Following stimulation, cells were stained for CD8, IFNγ, and TNFα. Data shown are pregated on live CD8 + CTL. The percent inhibition refers to loss of the effector function noted in the legend. (A) Representative flow plots. (B) Averaged data ± SEM from eight independent experiments assessed using two independently generated NP-specific CTL lines.

    Journal: Frontiers in Immunology

    Article Title: A Novel Function for the Streptococcus pneumoniae Aminopeptidase N: Inhibition of T Cell Effector Function through Regulation of TCR Signaling

    doi: 10.3389/fimmu.2017.01610

    Figure Lengend Snippet: Treatment of NP-specific CD8 + cytotoxic T lymphocyte (CTL) with EF3030-derived lysate inhibits the production of IFNγ and tumor necrosis factor α (TNFα) and the release of cytolytic granules. NP 147–155 -specific CTL was stimulated with peptide in the presence of increasing amounts of Spn lysate. Anti-CD107a antibody was included in the stimulation phase to identify cells releasing lytic granules. Following stimulation, cells were stained for CD8, IFNγ, and TNFα. Data shown are pregated on live CD8 + CTL. The percent inhibition refers to loss of the effector function noted in the legend. (A) Representative flow plots. (B) Averaged data ± SEM from eight independent experiments assessed using two independently generated NP-specific CTL lines.

    Article Snippet: Cells were then fixed and permeabilized (Cytofix/Cytoperm kit, BD Biosciences) followed by incubation with antibodies specific for IFNγ (Biolegend) and in some cases TNFα (Biolegend).

    Techniques: CTL Assay, Derivative Assay, Staining, Inhibition, Flow Cytometry, Generated

    Identification of cytokines as responsible for enhancing human immunodeficiency virus type I (HIV-1) infection in unactivated CD4 + T-cells. (A) Unactivated CD4 + T-cells were stimulated with different combinations of cytokines for 72 h. Then, cells were infected and p24 antigen production was evaluated at days 4 and 7 post-infection. Each condition was compared with the corresponding RPMI condition (negative control). As a positive control, PHA stimulation was used. Percentage of living CD4 + T-cells (B) and percentage of infected (GFP + ) CD4 + T-cells (C) after stimulation with the denoted treatments are shown. Data represent mean ± SD from four independent donors evaluated in duplicate. Concentrations of cytokines used in these experiments corresponded to the average concentrations found in monocyte-derived macrophage (MDM) supernatants stimulated with 25 ng/ml macrophage migration inhibitory factor (MIF) (peak effect) as follows: 250 pg/ml IL-6, 9,000 pg/ml IL-8, 1,400 pg/ml TNF-α, and 20 pg/ml IL-1β. (D) Neutralization of IL-8, IL-6, IL-1 β, and TNFα biological activity with monoclonal neutralizing antibodies. Primary CD4 + T-cells were incubated with supernatants derived from the 25 ng/ml MIF-treated HIV-infected MDM neutralized previously with 18 µg/ml anti-IL-8, 20 ng/ml anti-IL-6, 2 µg/ml anti-IL-1β, and 2 µg/ml anti-TNFα antibodies. Non-neutralized and isotype control antibody conditions were tested for comparison. Also, RPMI and PHA controls were included. Viral production was evaluated at day 4 post-infection. Data were analyzed by one-way ANOVA followed by Dunnett’s post-test (all conditions versus the corresponding RMPI control) in (A) and Tukey’s post-test in (D) . * p

    Journal: Frontiers in Immunology

    Article Title: Interaction Between Macrophage Migration Inhibitory Factor and CD74 in Human Immunodeficiency Virus Type I Infected Primary Monocyte-Derived Macrophages Triggers the Production of Proinflammatory Mediators and Enhances Infection of Unactivated CD4+ T Cells

    doi: 10.3389/fimmu.2018.01494

    Figure Lengend Snippet: Identification of cytokines as responsible for enhancing human immunodeficiency virus type I (HIV-1) infection in unactivated CD4 + T-cells. (A) Unactivated CD4 + T-cells were stimulated with different combinations of cytokines for 72 h. Then, cells were infected and p24 antigen production was evaluated at days 4 and 7 post-infection. Each condition was compared with the corresponding RPMI condition (negative control). As a positive control, PHA stimulation was used. Percentage of living CD4 + T-cells (B) and percentage of infected (GFP + ) CD4 + T-cells (C) after stimulation with the denoted treatments are shown. Data represent mean ± SD from four independent donors evaluated in duplicate. Concentrations of cytokines used in these experiments corresponded to the average concentrations found in monocyte-derived macrophage (MDM) supernatants stimulated with 25 ng/ml macrophage migration inhibitory factor (MIF) (peak effect) as follows: 250 pg/ml IL-6, 9,000 pg/ml IL-8, 1,400 pg/ml TNF-α, and 20 pg/ml IL-1β. (D) Neutralization of IL-8, IL-6, IL-1 β, and TNFα biological activity with monoclonal neutralizing antibodies. Primary CD4 + T-cells were incubated with supernatants derived from the 25 ng/ml MIF-treated HIV-infected MDM neutralized previously with 18 µg/ml anti-IL-8, 20 ng/ml anti-IL-6, 2 µg/ml anti-IL-1β, and 2 µg/ml anti-TNFα antibodies. Non-neutralized and isotype control antibody conditions were tested for comparison. Also, RPMI and PHA controls were included. Viral production was evaluated at day 4 post-infection. Data were analyzed by one-way ANOVA followed by Dunnett’s post-test (all conditions versus the corresponding RMPI control) in (A) and Tukey’s post-test in (D) . * p

    Article Snippet: A CD74 blocking antibody (BD Pharmingen, clone LN2), the recombinant human cytokines IL-6, IL-8, IL-1β (BioLegend), and TNFα (MiltenyiBiotec), and the cytokine neutralizing antibodies anti-IL-8 (R & D Systems), anti-IL-6, anti-IL-1β, and anti-TNFα (BioLegend) were obtained.

    Techniques: Infection, Negative Control, Positive Control, Derivative Assay, Migration, Neutralization, Activity Assay, Incubation

    BV6 does not induce NF-κB nuclear translocation SW620 cells were either untreated (a), treated with TNFα (b, 100 U/ml), BV6 (c, 5 μM) or both TNFα and BV6 (d) for 60 min. Cells were fixed, permeabilized, and stained with p65-specific antibody, followed by fluorescent dye-conjugated 2 nd antibody. The images were obtained with a confocal microscope. a1-d1 are low amplification images and a2-d2 are high amplification images.

    Journal: Oncotarget

    Article Title: NF-κB functions as a molecular link between tumor cells and Th1/Tc1 T cells in the tumor microenvironment to exert radiation-mediated tumor suppression

    doi: 10.18632/oncotarget.8246

    Figure Lengend Snippet: BV6 does not induce NF-κB nuclear translocation SW620 cells were either untreated (a), treated with TNFα (b, 100 U/ml), BV6 (c, 5 μM) or both TNFα and BV6 (d) for 60 min. Cells were fixed, permeabilized, and stained with p65-specific antibody, followed by fluorescent dye-conjugated 2 nd antibody. The images were obtained with a confocal microscope. a1-d1 are low amplification images and a2-d2 are high amplification images.

    Article Snippet: TNFα neutralization Tumor cells were irradiated and then cultured in the presence of BV6 plus either IgG (20 μg/ml) or anti-TNFα mAb (Biolegend, 20 μg/ml) for 24h.

    Techniques: Translocation Assay, Staining, Microscopy, Amplification

    Radiation-induced NF-κB directly regulates TNFα transcription that acts in concert with BV6 to induce tumor cell death A. The human TNFα promoter structure. The three NF-κB-binding consensus sequence elements are indicated at the bottom of the bar. B. Tumor cells were irradiated at a dose of 50 Gy and then cultured for 90 min. Nuclear extracts were prepared and analyzed for NF-κB activation using EMSA with the three DNA probes of the human TNFα promoter as indicated in A. C. Tumor cells were irradiated with the indicated dose and cultured for 24h. Cells were then analyzed for human TNFα mRNA level using quantitative PCR. D. Tumor cells were treated as indicated for 24h and analyzed for cell death using PI staining and flow cytometry analysis. E. Tumor cells were irradiated and then cultured in the presence of BV6 (5 μM). IgG or anti-TNFα neutralization antibody (200 μg/ml) was added to the culture, respectively, for 24h. Cell death was analyzed by PI staining and flow cytometry analysis. * p

    Journal: Oncotarget

    Article Title: NF-κB functions as a molecular link between tumor cells and Th1/Tc1 T cells in the tumor microenvironment to exert radiation-mediated tumor suppression

    doi: 10.18632/oncotarget.8246

    Figure Lengend Snippet: Radiation-induced NF-κB directly regulates TNFα transcription that acts in concert with BV6 to induce tumor cell death A. The human TNFα promoter structure. The three NF-κB-binding consensus sequence elements are indicated at the bottom of the bar. B. Tumor cells were irradiated at a dose of 50 Gy and then cultured for 90 min. Nuclear extracts were prepared and analyzed for NF-κB activation using EMSA with the three DNA probes of the human TNFα promoter as indicated in A. C. Tumor cells were irradiated with the indicated dose and cultured for 24h. Cells were then analyzed for human TNFα mRNA level using quantitative PCR. D. Tumor cells were treated as indicated for 24h and analyzed for cell death using PI staining and flow cytometry analysis. E. Tumor cells were irradiated and then cultured in the presence of BV6 (5 μM). IgG or anti-TNFα neutralization antibody (200 μg/ml) was added to the culture, respectively, for 24h. Cell death was analyzed by PI staining and flow cytometry analysis. * p

    Article Snippet: TNFα neutralization Tumor cells were irradiated and then cultured in the presence of BV6 plus either IgG (20 μg/ml) or anti-TNFα mAb (Biolegend, 20 μg/ml) for 24h.

    Techniques: Binding Assay, Sequencing, Irradiation, Cell Culture, Activation Assay, Real-time Polymerase Chain Reaction, Staining, Flow Cytometry, Cytometry, Neutralization

    Radiation and TNFα induce apoptosis and necroptosis A. Tumor cells were irradiated (R) at a dose of 50 Gy and then cultured in the presence of BV6 (B, 5 μM) plus Z-VAD (Z, 10 μM), Nec-1 (10 μM), and NSA (2 μM), either alone or in combinations, for 24 hours. Cell death was determined by PI staining and flow cytometry analysis. Column: mean, bar: SD. B. Tumor cells were cultured in the presence of TNFα (T, 100 U/ml) and BV6 (B, 5 μM) plus Z-VAD (Z, 10 μM), Nec-1 (10 μM), and NSA (2 μM), either alone or in combinations, for 24h, and analyzed for cell death as in A. Column: mean; Bar: SD.

    Journal: Oncotarget

    Article Title: NF-κB functions as a molecular link between tumor cells and Th1/Tc1 T cells in the tumor microenvironment to exert radiation-mediated tumor suppression

    doi: 10.18632/oncotarget.8246

    Figure Lengend Snippet: Radiation and TNFα induce apoptosis and necroptosis A. Tumor cells were irradiated (R) at a dose of 50 Gy and then cultured in the presence of BV6 (B, 5 μM) plus Z-VAD (Z, 10 μM), Nec-1 (10 μM), and NSA (2 μM), either alone or in combinations, for 24 hours. Cell death was determined by PI staining and flow cytometry analysis. Column: mean, bar: SD. B. Tumor cells were cultured in the presence of TNFα (T, 100 U/ml) and BV6 (B, 5 μM) plus Z-VAD (Z, 10 μM), Nec-1 (10 μM), and NSA (2 μM), either alone or in combinations, for 24h, and analyzed for cell death as in A. Column: mean; Bar: SD.

    Article Snippet: TNFα neutralization Tumor cells were irradiated and then cultured in the presence of BV6 plus either IgG (20 μg/ml) or anti-TNFα mAb (Biolegend, 20 μg/ml) for 24h.

    Techniques: Irradiation, Cell Culture, Staining, Flow Cytometry, Cytometry

    BV6 activates the alternate but not the canonical NF-κB A. The indicated human sarcoma cells were treated with BV6 (5 μM) for the indicated time. Cells were then analyzed by Western blotting analysis for p100/p52, pIκBα, and IκBα. B. The indicated human colon carcinoma cells were treated with BV6 (5 μM) for the indicated time and then analyzed for p100/p52, pIκBα, and IκBα as in A. C. The indicated human sarcoma cells were treated with BV6 (5μM, 1 and 2h), TNFα (100 U/ml, 1h), or both as indicated. Nuclear extracts were prepared from the tumor cells and analyzed for canonical NF-κB activity using EMSA with NF-κB consensus sequence-containing DNA probes (Santa Cruz Biotech). Anti-p65 and anti-p50 antibodies were used to identify the canonical NF-κB-DNA complexes. D. SW620 cells were treated with BV6 (5 μM, for 0.5, 1, and 2h) and TNFα (1 h) and analyzed for NF-κB activation as in C.

    Journal: Oncotarget

    Article Title: NF-κB functions as a molecular link between tumor cells and Th1/Tc1 T cells in the tumor microenvironment to exert radiation-mediated tumor suppression

    doi: 10.18632/oncotarget.8246

    Figure Lengend Snippet: BV6 activates the alternate but not the canonical NF-κB A. The indicated human sarcoma cells were treated with BV6 (5 μM) for the indicated time. Cells were then analyzed by Western blotting analysis for p100/p52, pIκBα, and IκBα. B. The indicated human colon carcinoma cells were treated with BV6 (5 μM) for the indicated time and then analyzed for p100/p52, pIκBα, and IκBα as in A. C. The indicated human sarcoma cells were treated with BV6 (5μM, 1 and 2h), TNFα (100 U/ml, 1h), or both as indicated. Nuclear extracts were prepared from the tumor cells and analyzed for canonical NF-κB activity using EMSA with NF-κB consensus sequence-containing DNA probes (Santa Cruz Biotech). Anti-p65 and anti-p50 antibodies were used to identify the canonical NF-κB-DNA complexes. D. SW620 cells were treated with BV6 (5 μM, for 0.5, 1, and 2h) and TNFα (1 h) and analyzed for NF-κB activation as in C.

    Article Snippet: TNFα neutralization Tumor cells were irradiated and then cultured in the presence of BV6 plus either IgG (20 μg/ml) or anti-TNFα mAb (Biolegend, 20 μg/ml) for 24h.

    Techniques: Western Blot, Activity Assay, Sequencing, Activation Assay

    Model of radiation modulation of NF-κB and CTL anti-tumor immune response in the tumor microenvironment A. Radiation initiates at least four molecular effects: 1) radiation activates the canonical NF-κB p65/p50 and p50/p50 complexes that directly bind to the TNFα promoter to activate its transcription; 2) radiation-activated canonical NF-κB p65/p50 and p50/p50 complexes also directly bind to the FAS promoter to activate its transcription to increase tumor cell sensitivity to apoptosis induction by FasL of tumor-infiltrating Tc1 cells; 3) radiation-activated NF-κB also up-regulates the T cell chemokines CCL2 and CCL5 to attract T cell infiltration to the tumor microenvironment; and 4) radiation-generated dsDNA fragments activate the STING pathway, as characterized by increased IRF3 and IFNβ expression, to induce T cell activation. B. TNFα in the tumor microenvironment induces tumor apoptosis and necroptosis in an autocrine manner, and RIP1 is a key molecular switch that determines whether TNFα induces cell death or survival signaling pathways. cIAP1 and cIAP2 ubiquitylates RIP1 in the TNFR cytoplasmic complex (complex I) and the ubiquitylated RIP1 promotes cell survival by preventing the formation of the death complex IIb. BV6 induces cIAP1 and cIAP2 degradation. Degradation of cIAP1 and cIAP2 leads to activation of caspase 9. Degradation of cIAP1 and cIAP2 also leads to deubiquitylation of RIP1. Deubiquitylated RIP1 dissociates from the membrane-bound TNFR complex I to form cytosolic complex IIb that mediates both caspase-dependent apoptosis and RIP3-dependent necroptosis.

    Journal: Oncotarget

    Article Title: NF-κB functions as a molecular link between tumor cells and Th1/Tc1 T cells in the tumor microenvironment to exert radiation-mediated tumor suppression

    doi: 10.18632/oncotarget.8246

    Figure Lengend Snippet: Model of radiation modulation of NF-κB and CTL anti-tumor immune response in the tumor microenvironment A. Radiation initiates at least four molecular effects: 1) radiation activates the canonical NF-κB p65/p50 and p50/p50 complexes that directly bind to the TNFα promoter to activate its transcription; 2) radiation-activated canonical NF-κB p65/p50 and p50/p50 complexes also directly bind to the FAS promoter to activate its transcription to increase tumor cell sensitivity to apoptosis induction by FasL of tumor-infiltrating Tc1 cells; 3) radiation-activated NF-κB also up-regulates the T cell chemokines CCL2 and CCL5 to attract T cell infiltration to the tumor microenvironment; and 4) radiation-generated dsDNA fragments activate the STING pathway, as characterized by increased IRF3 and IFNβ expression, to induce T cell activation. B. TNFα in the tumor microenvironment induces tumor apoptosis and necroptosis in an autocrine manner, and RIP1 is a key molecular switch that determines whether TNFα induces cell death or survival signaling pathways. cIAP1 and cIAP2 ubiquitylates RIP1 in the TNFR cytoplasmic complex (complex I) and the ubiquitylated RIP1 promotes cell survival by preventing the formation of the death complex IIb. BV6 induces cIAP1 and cIAP2 degradation. Degradation of cIAP1 and cIAP2 leads to activation of caspase 9. Degradation of cIAP1 and cIAP2 also leads to deubiquitylation of RIP1. Deubiquitylated RIP1 dissociates from the membrane-bound TNFR complex I to form cytosolic complex IIb that mediates both caspase-dependent apoptosis and RIP3-dependent necroptosis.

    Article Snippet: TNFα neutralization Tumor cells were irradiated and then cultured in the presence of BV6 plus either IgG (20 μg/ml) or anti-TNFα mAb (Biolegend, 20 μg/ml) for 24h.

    Techniques: CTL Assay, Generated, Expressing, Activation Assay

    TNFα and BV6 cooperate to activate sequential necroptosis and apoptosis A. Tumor cells were cultured in the presence of TNFα (100 U/ml), BV6 (5 μM), or both for the indicated time and analyzed by Western blotting analysis for the indicated caspases. PARP was used as an apoptosis indicator. B. Tumor cells were cultured in the presence of TNFα (100 U/ml), BV6 (5 μM), plus Z-VAD (10 μM), Nec-1 (10 μM), or NSA (2 μM), respectively for 4 h, and analyzed by Western blotting for the indicated caspases and PARP.

    Journal: Oncotarget

    Article Title: NF-κB functions as a molecular link between tumor cells and Th1/Tc1 T cells in the tumor microenvironment to exert radiation-mediated tumor suppression

    doi: 10.18632/oncotarget.8246

    Figure Lengend Snippet: TNFα and BV6 cooperate to activate sequential necroptosis and apoptosis A. Tumor cells were cultured in the presence of TNFα (100 U/ml), BV6 (5 μM), or both for the indicated time and analyzed by Western blotting analysis for the indicated caspases. PARP was used as an apoptosis indicator. B. Tumor cells were cultured in the presence of TNFα (100 U/ml), BV6 (5 μM), plus Z-VAD (10 μM), Nec-1 (10 μM), or NSA (2 μM), respectively for 4 h, and analyzed by Western blotting for the indicated caspases and PARP.

    Article Snippet: TNFα neutralization Tumor cells were irradiated and then cultured in the presence of BV6 plus either IgG (20 μg/ml) or anti-TNFα mAb (Biolegend, 20 μg/ml) for 24h.

    Techniques: Cell Culture, Western Blot

    Cell-mediated immune responses. (A) Gating strategy for analysis of CD4+ and CD8+ T cell cytokine secretion without stimulus or after stimulation with HBsAg peptide(B) Percentage of HBV-specific Granzyme-, IFNγ-, and TNFα-secreting CD8+ T-cells and HBV-specific IL-2-secreting CD4+ T-cells analyzed in Responder and Non-Responder HIV-infected individuals at baseline and in response to HBV vaccine booster dose. * p

    Journal: PLoS ONE

    Article Title: Humoral and cell-mediated immune responses after a booster dose of HBV vaccine in HIV-infected children, adolescents and young adults

    doi: 10.1371/journal.pone.0192638

    Figure Lengend Snippet: Cell-mediated immune responses. (A) Gating strategy for analysis of CD4+ and CD8+ T cell cytokine secretion without stimulus or after stimulation with HBsAg peptide(B) Percentage of HBV-specific Granzyme-, IFNγ-, and TNFα-secreting CD8+ T-cells and HBV-specific IL-2-secreting CD4+ T-cells analyzed in Responder and Non-Responder HIV-infected individuals at baseline and in response to HBV vaccine booster dose. * p

    Article Snippet: The following antibodies were used: anti-human CD4-PeCy5 (eBioscience, USA), anti-human CD8-PC5 (Beckman Coulter, California, USA), anti-human IL-2-PE (eBioscience, USA), anti-human Granzyme B-PE (R & D Systems, Minneapolis, USA), anti-human IFNγ-FITC (eBioscience, USA), anti-human CCR7-PE (R & D Systems, Minneapolis, USA) and anti-human TNFα-PE (Biolegend, USA).

    Techniques: Infection

    Effects of stress and resveratrol treatment on splenic TNFα levels 5 days post social defeat or control exposure RSV decreased expression of the pro-inflammatory cytokine TNFα following Con-A stimulation in splenic cells of passive coping rats. Data are expressed as the percentage of total CD4+ cells expressing TNFα. (A, B, C) Data shown are flow cytometry dot plots from one representative animal in each group shown. (D) Graph of combined data from all experiments (n=8 [control + vehicle]; n=7 [passive + vehicle]; n=8 [active + vehicle]; n=5 [control + 10mg/kg RSV]; n=8 [passive + 10mg/kg RSV]; n=4 [active + 10mg/kg RSV]; n=5 [control + 30mg/kg RSV]; n=9 [passive + 30mg/kg RSV]; n=4 [active + 30mg/kg RSV]). Fisher’s LSD post-hoc analysis revealed statistical significance (ƒ p

    Journal: Brain, behavior, and immunity

    Article Title: The protective effects of resveratrol on social stress-induced cytokine release and depressive-like behavior

    doi: 10.1016/j.bbi.2016.08.019

    Figure Lengend Snippet: Effects of stress and resveratrol treatment on splenic TNFα levels 5 days post social defeat or control exposure RSV decreased expression of the pro-inflammatory cytokine TNFα following Con-A stimulation in splenic cells of passive coping rats. Data are expressed as the percentage of total CD4+ cells expressing TNFα. (A, B, C) Data shown are flow cytometry dot plots from one representative animal in each group shown. (D) Graph of combined data from all experiments (n=8 [control + vehicle]; n=7 [passive + vehicle]; n=8 [active + vehicle]; n=5 [control + 10mg/kg RSV]; n=8 [passive + 10mg/kg RSV]; n=4 [active + 10mg/kg RSV]; n=5 [control + 30mg/kg RSV]; n=9 [passive + 30mg/kg RSV]; n=4 [active + 30mg/kg RSV]). Fisher’s LSD post-hoc analysis revealed statistical significance (ƒ p

    Article Snippet: Primary antibodies used to label inflammatory cytokines were PE-anti rat TNFα (Biolegend, San Diego CA), Biotin anti-rat IL-1β (Abcam, Cambridge MA), mouse anti-rat IL-6 (Invitrogen, Camarillo CA), and PE-anti-rat IL-10 (BD Biosciences, San Jose CA) or appropriate isotype controls.

    Techniques: Expressing, Flow Cytometry, Cytometry

    Splenic CD4 + forkhead box protein 3 (FoxP3) + regulatory T cells (T reg ) from FoxP3 GFP /B6 mice [wild-type (WT) T reg ] successfully suppresses inflammatory cytokine production by CD8 T cells. (a) Intracellular staining analysis for interferon (IFN)-γ and tumour necrosis factor (TNF)-α-expressing CD8 T cells in liver. (b) Level of IFN-γ, TNF-α and interleukin (IL)-6 in liver protein extracts from recipients. (c) Serum levels of IFN-γ, TNF-α, IL12/IL23p40 and IL-6 in recipient mice. dnRII CD8 group ( n = 11), dnRII CD8 + dnRII T reg group ( n = 7) and dnRII CD8 + WT T reg group ( n = 7). Data are expressed as mean ± standard error of the mean. * P

    Journal: Clinical and Experimental Immunology

    Article Title: Successful immunotherapy of autoimmune cholangitis by adoptive transfer of forkhead box protein 3+ regulatory T cells

    doi: 10.1111/cei.12415

    Figure Lengend Snippet: Splenic CD4 + forkhead box protein 3 (FoxP3) + regulatory T cells (T reg ) from FoxP3 GFP /B6 mice [wild-type (WT) T reg ] successfully suppresses inflammatory cytokine production by CD8 T cells. (a) Intracellular staining analysis for interferon (IFN)-γ and tumour necrosis factor (TNF)-α-expressing CD8 T cells in liver. (b) Level of IFN-γ, TNF-α and interleukin (IL)-6 in liver protein extracts from recipients. (c) Serum levels of IFN-γ, TNF-α, IL12/IL23p40 and IL-6 in recipient mice. dnRII CD8 group ( n = 11), dnRII CD8 + dnRII T reg group ( n = 7) and dnRII CD8 + WT T reg group ( n = 7). Data are expressed as mean ± standard error of the mean. * P

    Article Snippet: The cells were stained for surface CD8α and TCR-β, fixed and permeabilized with BD Cytofix/Cytoperm Solution (BD Biosciences, San Diego, CA, USA), then stained for intracellular interferon (IFN)-γ (eBioscience), cytotoxic T lymphocyte antigen-4 (CTLA-4), Helios, IL-10 and tumour necrosis factor (TNF)-α (BioLegend) [ ].

    Techniques: Mouse Assay, Staining, Expressing

    HIF1α is critical for pro-inflammatory macrophage differentiation in vitro . Sorted PEMs from WT and HIF1α −/− mice were stimulated with LPS (100 ng/mL) or curdlan (100 ng/mL) for 10–12 h, and the indicated mRNA expression was determined with qPCR ( A ). Supernatant was collected and the concentration of the indicated cytokines was determined with an ELISA ( B ). ( C ) Peritoneal exudate cells from WT and HIF1α −/− mice were activated with the indicated stimuli, and the intracellular expression of TNFα in F4/80 + macrophages was determined with flow cytometry; a representative image is shown in the left figure, and data are summarized in the right figure. ( D ) Sorted PEMs were stimulated with LPS or curdlan for 10–12 h, and the mRNA expression of glycolytic pathway associated molecules was determined with qPCR. Data are presented as the means ± SD (n = 3–5). One representative experiment of three independent experiments is shown. * P

    Journal: Scientific Reports

    Article Title: HIF1α-dependent glycolysis promotes macrophage functional activities in protecting against bacterial and fungal infection

    doi: 10.1038/s41598-018-22039-9

    Figure Lengend Snippet: HIF1α is critical for pro-inflammatory macrophage differentiation in vitro . Sorted PEMs from WT and HIF1α −/− mice were stimulated with LPS (100 ng/mL) or curdlan (100 ng/mL) for 10–12 h, and the indicated mRNA expression was determined with qPCR ( A ). Supernatant was collected and the concentration of the indicated cytokines was determined with an ELISA ( B ). ( C ) Peritoneal exudate cells from WT and HIF1α −/− mice were activated with the indicated stimuli, and the intracellular expression of TNFα in F4/80 + macrophages was determined with flow cytometry; a representative image is shown in the left figure, and data are summarized in the right figure. ( D ) Sorted PEMs were stimulated with LPS or curdlan for 10–12 h, and the mRNA expression of glycolytic pathway associated molecules was determined with qPCR. Data are presented as the means ± SD (n = 3–5). One representative experiment of three independent experiments is shown. * P

    Article Snippet: For intracellular staining, TNFα expression (MP6-XT22; Biolegend) was analyzed by flow cytometry according to the manufacturer’s instructions.

    Techniques: In Vitro, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry

    HIF1α is required for pro-inflammatory macrophage differentiation following Listeria bacterial infection. C57BL/6 WT or HIF1α −/− mice were i.v. injected with 1 × 10 5 CFU of L. monocytogenes bacteria. 48 h after infection, mouse livers were collected, and the CFU were determined ( A ). Infected mice developed severe infection and inflammatory cell infiltration, as shown by the histological staining of H E ( B ). At the same time point after infection, serum or peritoneal exudate TNFα levels were determined using an ELISA ( C and E ); the sorted splenic macrophages or PEMs were stimulated with LPS for 12 h, the supernatant was collected, and TNFα levels were determined using an ELISA ( D and F ); the glycolytic pathway activity of macrophages was also determined ( H and I ). TNFα expression in F4/80 + macrophages from peritoneal exudates was analyzed with FCM. A representative figure is shown in the left image, and the data are summarized in the right image ( G ). Data are presented as the means ± SD (n = 3–5). One representative experiment of three independent experiments is shown. *** P

    Journal: Scientific Reports

    Article Title: HIF1α-dependent glycolysis promotes macrophage functional activities in protecting against bacterial and fungal infection

    doi: 10.1038/s41598-018-22039-9

    Figure Lengend Snippet: HIF1α is required for pro-inflammatory macrophage differentiation following Listeria bacterial infection. C57BL/6 WT or HIF1α −/− mice were i.v. injected with 1 × 10 5 CFU of L. monocytogenes bacteria. 48 h after infection, mouse livers were collected, and the CFU were determined ( A ). Infected mice developed severe infection and inflammatory cell infiltration, as shown by the histological staining of H E ( B ). At the same time point after infection, serum or peritoneal exudate TNFα levels were determined using an ELISA ( C and E ); the sorted splenic macrophages or PEMs were stimulated with LPS for 12 h, the supernatant was collected, and TNFα levels were determined using an ELISA ( D and F ); the glycolytic pathway activity of macrophages was also determined ( H and I ). TNFα expression in F4/80 + macrophages from peritoneal exudates was analyzed with FCM. A representative figure is shown in the left image, and the data are summarized in the right image ( G ). Data are presented as the means ± SD (n = 3–5). One representative experiment of three independent experiments is shown. *** P

    Article Snippet: For intracellular staining, TNFα expression (MP6-XT22; Biolegend) was analyzed by flow cytometry according to the manufacturer’s instructions.

    Techniques: Infection, Mouse Assay, Injection, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Expressing

    Glycolytic pathway activity was required for pro-inflammatory macrophage differentiation. PEMs with indicated treatments for 10–12 h (2-DG, 1 mmol/L; LPS, 100 ng/mL; curdlan, 100 ng/mL; A – D ). A . HIF1α mRNA expression of PEMs was determined with qPCR (Value of control groups was set to 1). ( B and C) Supernatants were collected, and the indicated cytokine concentration was determined using an ELISA. ( D ) The glycolytic pathway activity was summarized. Mice were i.p. injected with 1 × 10 5 of C. albicans yeast and also injected intraperitoneally with 2-DG (2 g/kg body weight) or solvent alone (PBS; Ctrl) for 9 days. 2-DG or PBS were given daily up until the day before the mice were euthanized ( E – H ). ( E ) Mouse liver and kidneys were collected, and the CFU was evaluated. ( F ) Serum TNFα concentration ( F ). ( G ) TNFα expression of PEMs with FCM. A representative image is shown in the left figure and data are summarized in the right figure. ( H) The glycolytic pathway activity of PEMs. Data are presented as the means ± SD (n = 3–4). One representative experiment of three independent experiments is shown. P

    Journal: Scientific Reports

    Article Title: HIF1α-dependent glycolysis promotes macrophage functional activities in protecting against bacterial and fungal infection

    doi: 10.1038/s41598-018-22039-9

    Figure Lengend Snippet: Glycolytic pathway activity was required for pro-inflammatory macrophage differentiation. PEMs with indicated treatments for 10–12 h (2-DG, 1 mmol/L; LPS, 100 ng/mL; curdlan, 100 ng/mL; A – D ). A . HIF1α mRNA expression of PEMs was determined with qPCR (Value of control groups was set to 1). ( B and C) Supernatants were collected, and the indicated cytokine concentration was determined using an ELISA. ( D ) The glycolytic pathway activity was summarized. Mice were i.p. injected with 1 × 10 5 of C. albicans yeast and also injected intraperitoneally with 2-DG (2 g/kg body weight) or solvent alone (PBS; Ctrl) for 9 days. 2-DG or PBS were given daily up until the day before the mice were euthanized ( E – H ). ( E ) Mouse liver and kidneys were collected, and the CFU was evaluated. ( F ) Serum TNFα concentration ( F ). ( G ) TNFα expression of PEMs with FCM. A representative image is shown in the left figure and data are summarized in the right figure. ( H) The glycolytic pathway activity of PEMs. Data are presented as the means ± SD (n = 3–4). One representative experiment of three independent experiments is shown. P

    Article Snippet: For intracellular staining, TNFα expression (MP6-XT22; Biolegend) was analyzed by flow cytometry according to the manufacturer’s instructions.

    Techniques: Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Mouse Assay, Injection

    Pharmacologically targeting HIF1α and glycolytic pathway activity in mouse and human cells. Mouse PEMs ( A – E ) or human macrophages ( F – J ) pulsed with C. albicans yeast (1 × 10 5 ) for 3 days in the absence or presence of CoCl 2 (200 µM), 2-ME (2 µM), or 2-DG (1 mmol/L). ( A and E) The HIF1α mRNA expression of macrophages was determined with qPCR. ( B and F ) The TNFα concentration of supernatants was determined. ( C and G ) The CFU was evaluated. ( D and H) The Glut1 mRNA expression was determined with qPCR. Data are presented as the means ± SD (n = 3–4). One representative experiment of three independent experiments is shown. * P

    Journal: Scientific Reports

    Article Title: HIF1α-dependent glycolysis promotes macrophage functional activities in protecting against bacterial and fungal infection

    doi: 10.1038/s41598-018-22039-9

    Figure Lengend Snippet: Pharmacologically targeting HIF1α and glycolytic pathway activity in mouse and human cells. Mouse PEMs ( A – E ) or human macrophages ( F – J ) pulsed with C. albicans yeast (1 × 10 5 ) for 3 days in the absence or presence of CoCl 2 (200 µM), 2-ME (2 µM), or 2-DG (1 mmol/L). ( A and E) The HIF1α mRNA expression of macrophages was determined with qPCR. ( B and F ) The TNFα concentration of supernatants was determined. ( C and G ) The CFU was evaluated. ( D and H) The Glut1 mRNA expression was determined with qPCR. Data are presented as the means ± SD (n = 3–4). One representative experiment of three independent experiments is shown. * P

    Article Snippet: For intracellular staining, TNFα expression (MP6-XT22; Biolegend) was analyzed by flow cytometry according to the manufacturer’s instructions.

    Techniques: Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Concentration Assay

    LPS induces miR-155 and TNFα expression in hepatic immune cells. Isolated LMNCs and Kupffer cells from C57Bl/6 WT mice were stimulated or not with 100ng/ml LPS for 6 hours in vitro. miR-155 expression (A: LMNCs, C: KCs) and TNF-α protein secretion (B: LMNCs, D: KCs) were measured in the cells and in the supernatant, respectively (n = 8-10/group). RNA was isolated from C57Bl/6 WT mice fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 3, 6 and 8 weeks. miR155 and TNFα mRNA expression was determined (n = 5-6/group, total 32) and correlation was plotted (E); correlation coefficient is shown. (*) indicates p

    Journal: PLoS ONE

    Article Title: MicroRNA-155 Deficiency Attenuates Liver Steatosis and Fibrosis without Reducing Inflammation in a Mouse Model of Steatohepatitis

    doi: 10.1371/journal.pone.0129251

    Figure Lengend Snippet: LPS induces miR-155 and TNFα expression in hepatic immune cells. Isolated LMNCs and Kupffer cells from C57Bl/6 WT mice were stimulated or not with 100ng/ml LPS for 6 hours in vitro. miR-155 expression (A: LMNCs, C: KCs) and TNF-α protein secretion (B: LMNCs, D: KCs) were measured in the cells and in the supernatant, respectively (n = 8-10/group). RNA was isolated from C57Bl/6 WT mice fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 3, 6 and 8 weeks. miR155 and TNFα mRNA expression was determined (n = 5-6/group, total 32) and correlation was plotted (E); correlation coefficient is shown. (*) indicates p

    Article Snippet: Serum and liver TNFα (BioLegend Inc ., San Diego CA , USA) , IL-1β (R&D Systems , Minneapolis , MN , USA) and MCP1 (BioLegend Inc ., San Diego CA , USA) levels were determined by ELISA as described by manufactures.

    Techniques: Expressing, Isolation, Mouse Assay, In Vitro

    miR-155 deficiency does not attenuate hepatic inflammation in MCD-steatohepatitis. Wild type (WT) and miR-155 deficient (KO) mice were fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 5 weeks. Liver TNFα (A top panel: mRNA, A bottom panel: protein), MCP1 (B top panel: mRNA, B bottom panel: protein) and IL1-β (C top panel: mRNA, C bottom panel: protein) mRNA and protein levels were measured by qPCR and ELISA, respectively (n = 6-8/group). NF-κB nuclear binding was evaluated by EMSA using liver nuclear extracts (D, top panel: representative blot, bottom panel: densitometry showing cumulative data of n = 6/group). (*) indicates p

    Journal: PLoS ONE

    Article Title: MicroRNA-155 Deficiency Attenuates Liver Steatosis and Fibrosis without Reducing Inflammation in a Mouse Model of Steatohepatitis

    doi: 10.1371/journal.pone.0129251

    Figure Lengend Snippet: miR-155 deficiency does not attenuate hepatic inflammation in MCD-steatohepatitis. Wild type (WT) and miR-155 deficient (KO) mice were fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 5 weeks. Liver TNFα (A top panel: mRNA, A bottom panel: protein), MCP1 (B top panel: mRNA, B bottom panel: protein) and IL1-β (C top panel: mRNA, C bottom panel: protein) mRNA and protein levels were measured by qPCR and ELISA, respectively (n = 6-8/group). NF-κB nuclear binding was evaluated by EMSA using liver nuclear extracts (D, top panel: representative blot, bottom panel: densitometry showing cumulative data of n = 6/group). (*) indicates p

    Article Snippet: Serum and liver TNFα (BioLegend Inc ., San Diego CA , USA) , IL-1β (R&D Systems , Minneapolis , MN , USA) and MCP1 (BioLegend Inc ., San Diego CA , USA) levels were determined by ELISA as described by manufactures.

    Techniques: Mouse Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Binding Assay

    MCD diet-induced steatohepatitis is associated with increased miR-155 expression in parenchymal and non-parenchymal cells. C57Bl/6 mice were fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 3, 6 and 8 weeks. Serum alanine aminotransferase levels (A) and TNFα mRNA expression (B) were determined (n = 5-8/group). Liver fibrosis was assessed by Sirius Red staining (100x; n = 3-6/group), representative slides are shown (C). miR-155 expression was detected by qPCR in total livers (n = 5-8/group) (D). Primary murine hepatocytes (n = 7/group), Kupffer cells (n = 6, pooled data, 2 datapoints/group) and liver mononuclear cells (LMNC; n = 3-4/group) were isolated from a subset of mice after 6 weeks of MCS or MCD diet feeding and cell-specific miR-155 expression was determined and represented as 1/dCt (E). miR-155* expression was determined in total livers (F). (*) indicates p

    Journal: PLoS ONE

    Article Title: MicroRNA-155 Deficiency Attenuates Liver Steatosis and Fibrosis without Reducing Inflammation in a Mouse Model of Steatohepatitis

    doi: 10.1371/journal.pone.0129251

    Figure Lengend Snippet: MCD diet-induced steatohepatitis is associated with increased miR-155 expression in parenchymal and non-parenchymal cells. C57Bl/6 mice were fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 3, 6 and 8 weeks. Serum alanine aminotransferase levels (A) and TNFα mRNA expression (B) were determined (n = 5-8/group). Liver fibrosis was assessed by Sirius Red staining (100x; n = 3-6/group), representative slides are shown (C). miR-155 expression was detected by qPCR in total livers (n = 5-8/group) (D). Primary murine hepatocytes (n = 7/group), Kupffer cells (n = 6, pooled data, 2 datapoints/group) and liver mononuclear cells (LMNC; n = 3-4/group) were isolated from a subset of mice after 6 weeks of MCS or MCD diet feeding and cell-specific miR-155 expression was determined and represented as 1/dCt (E). miR-155* expression was determined in total livers (F). (*) indicates p

    Article Snippet: Serum and liver TNFα (BioLegend Inc ., San Diego CA , USA) , IL-1β (R&D Systems , Minneapolis , MN , USA) and MCP1 (BioLegend Inc ., San Diego CA , USA) levels were determined by ELISA as described by manufactures.

    Techniques: Expressing, Mouse Assay, Staining, Real-time Polymerase Chain Reaction, Isolation

    Role of neutrophils and monocytes/macrophages in DAH A , peritoneal exudate cells (PEC) and bone marrow (BM) neutrophils from wild type mice treated 14-d earlier with pristane, with or without anti-Ly6G depletion (mAb IA8), were analyzed by flow cytometry. Left , surface staining with anti-Ly6G (mAb RB6-8C5)-APC-Cy7 and anti-CD11b-Pacific blue showing depletion of neutrophils (box) from the peritoneum but not bone marrow. Right , staining with anti-Ly6G (mAb RB6-8C5)-PE (surface stain) and anti-TNFα-APC (intracellular stain) showing loss of intracellular TNFα + peritoneal, but not bone marrow, Ly6G + cells. B , neutrophil depletion with anti-Ly6G antibodies. Wild type (WT) mice were treated with anti-Ly6G mAb 1-d before and 7-d after pristane treatment or left untreated (Control) (n = 6 per group). H E staining showed perivascular inflammatory cell infiltrates in controls, but not anti-Ly6G-treated mice. Efficacy of neutrophil depletion in lung was verified by staining with anti-neutrophil elastase antibodies ( right ). C , anti-F4/80 staining of macrophages in WT mice after depleting neutrophils with anti-Ly6G antibodies. F4/80 + cells are indicated by arrows. D , morphometric quantification of neutrophil elastase staining in lung from control vs. anti-Ly6G-treated mice. E , H E staining ( left ) and gross pathology ( right ) of lungs (14-d after pristane) from WT mice either treated with clodronate liposomes (CloLip, n = 8) 1-d before and 7-d after pristane or left untreated (No Rx, n = 6). Perivascular inflammatory cell infiltrates and DAH were absent after CloLip treatment. F , frequency of DAH in wild type (WT) and elastase-deficient (Ela−/−) mice 14-d after pristane treatment and in WT mice treated with anti-Ly6G antibodies (Ly6G) or with CloLip 1-d before and 7-d after pristane treatment. Pathology was assessed at 14-d. **, P

    Journal: Arthritis & rheumatology (Hoboken, N.J.)

    Article Title: Pathogenesis of diffuse alveolar hemorrhage in murine lupus

    doi: 10.1002/art.40077

    Figure Lengend Snippet: Role of neutrophils and monocytes/macrophages in DAH A , peritoneal exudate cells (PEC) and bone marrow (BM) neutrophils from wild type mice treated 14-d earlier with pristane, with or without anti-Ly6G depletion (mAb IA8), were analyzed by flow cytometry. Left , surface staining with anti-Ly6G (mAb RB6-8C5)-APC-Cy7 and anti-CD11b-Pacific blue showing depletion of neutrophils (box) from the peritoneum but not bone marrow. Right , staining with anti-Ly6G (mAb RB6-8C5)-PE (surface stain) and anti-TNFα-APC (intracellular stain) showing loss of intracellular TNFα + peritoneal, but not bone marrow, Ly6G + cells. B , neutrophil depletion with anti-Ly6G antibodies. Wild type (WT) mice were treated with anti-Ly6G mAb 1-d before and 7-d after pristane treatment or left untreated (Control) (n = 6 per group). H E staining showed perivascular inflammatory cell infiltrates in controls, but not anti-Ly6G-treated mice. Efficacy of neutrophil depletion in lung was verified by staining with anti-neutrophil elastase antibodies ( right ). C , anti-F4/80 staining of macrophages in WT mice after depleting neutrophils with anti-Ly6G antibodies. F4/80 + cells are indicated by arrows. D , morphometric quantification of neutrophil elastase staining in lung from control vs. anti-Ly6G-treated mice. E , H E staining ( left ) and gross pathology ( right ) of lungs (14-d after pristane) from WT mice either treated with clodronate liposomes (CloLip, n = 8) 1-d before and 7-d after pristane or left untreated (No Rx, n = 6). Perivascular inflammatory cell infiltrates and DAH were absent after CloLip treatment. F , frequency of DAH in wild type (WT) and elastase-deficient (Ela−/−) mice 14-d after pristane treatment and in WT mice treated with anti-Ly6G antibodies (Ly6G) or with CloLip 1-d before and 7-d after pristane treatment. Pathology was assessed at 14-d. **, P

    Article Snippet: In some cases, the cells were fixed, permeabilized with Perm/fix buffer (eBioscience, San Diego, CA), and intracellularly stained with anti-TNFα-allophycocyanin (APC) (BioLegend).

    Techniques: Mouse Assay, Flow Cytometry, Cytometry, Staining

    CD8 T-cell TNFα secretion after in vitro stimulation with JCV VP1 peptide. Circles indicate seven healthy control subjects; triangles indicate the patient’s samples. The dotted line indicates the detection threshold. Unstim = TNFα secretion of unstimulated CD8 T-cells; stim = TNFα secretion of CD8 T-cells stimulated with JCV VP1 peptide pool for 6 h.

    Journal: JMM Case Reports

    Article Title: Progressive multifocal leukoencephalopathy and black fungus in a patient with rheumatoid arthritis without severe lymphocytopenia

    doi: 10.1099/jmmcr.0.005053

    Figure Lengend Snippet: CD8 T-cell TNFα secretion after in vitro stimulation with JCV VP1 peptide. Circles indicate seven healthy control subjects; triangles indicate the patient’s samples. The dotted line indicates the detection threshold. Unstim = TNFα secretion of unstimulated CD8 T-cells; stim = TNFα secretion of CD8 T-cells stimulated with JCV VP1 peptide pool for 6 h.

    Article Snippet: In brief, thawed peripheral blood mononuclear cell (PBMC) was either rested for 2 h and incubated with 1 µg ml−1 brefeldin A for additional 4 h or stimulated for 6 h with 20 µl of PepTivator® stock solution for 5×106 PBMC/ml, as well as addition of 1 µg ml−1 brefeldin A after 2 h. Harvested cells were then stained with fluorochrome-conjugated antibodies against CD3, CD4, CD8 and CD56; fixated and permeabilized and intracellularly stained with a fluorochrome-conjugated antibody against TNFα (Biolegend) as previously described ( ).

    Techniques: In Vitro

    Increased TNFα surface expression and production in ADAM10 B−/− B cells

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Disturbed follicular architecture in B cell ADAM10 knockouts is mediated by compensatory increases in ADAM17 and TNFα shedding

    doi: 10.4049/jimmunol.1302042

    Figure Lengend Snippet: Increased TNFα surface expression and production in ADAM10 B−/− B cells

    Article Snippet: Kit reagents were prepared according to manufacturer’s protocol and tyramide amplification using the “Peroxidase Labeling assay” was performed with the following modifications: Cells were incubated with blocking reagent (10 μg anti-mouse unlabeled CD16/32 (2.4G2)) for 15 minutes; stained with biotin anti-mouse TNFα primary antibody (Biolegend), and following tyramide labeling, cells were washed twice and stained with anti-mouse B220 (see above) for 30 minutes and examined on a BD Canto Flow analyzer, data analysis was with FCS Express, v. 4.

    Techniques: Expressing

    Increased TNFα gene expression and message stability in ADAM10 B−/− B cells

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Disturbed follicular architecture in B cell ADAM10 knockouts is mediated by compensatory increases in ADAM17 and TNFα shedding

    doi: 10.4049/jimmunol.1302042

    Figure Lengend Snippet: Increased TNFα gene expression and message stability in ADAM10 B−/− B cells

    Article Snippet: Kit reagents were prepared according to manufacturer’s protocol and tyramide amplification using the “Peroxidase Labeling assay” was performed with the following modifications: Cells were incubated with blocking reagent (10 μg anti-mouse unlabeled CD16/32 (2.4G2)) for 15 minutes; stained with biotin anti-mouse TNFα primary antibody (Biolegend), and following tyramide labeling, cells were washed twice and stained with anti-mouse B220 (see above) for 30 minutes and examined on a BD Canto Flow analyzer, data analysis was with FCS Express, v. 4.

    Techniques: Expressing

    Tumor necrosis factor-α gene and protein expression in the 3 × Tg mouse. (A) Fold-change (2^(− δ δCt)) is the normalized gene expression (2^(−δ Ct)) in the test sample (3 × Tg treated with Thal or 3,6′-DT) divided by the normalized gene expression (2^(−δ Ct)) in the control sample (vehicle-treated 3 × Tg). Fold-change values of less than one indicate a negative- or downregulation. Both Thal and 3,6′-DT downregulated TNFα gene expression but the value was significant only in the 3,6′-DT group ( P = 0.033). (B) TNFα protein levels are doubled in the cortex of 3 × Tg mice compared with Non-Tg mice. 3,6′-DT, but not Thal, reduced TNFα protein levels near to Non-Tg levels in 3 × Tg mice. n = 5 to 8 per group. One-way analysis of variance ( P = 0.062, * P

    Journal: Journal of Neuroinflammation

    Article Title: Early intervention with a small molecule inhibitor for tumor nefosis factor-? prevents cognitive deficits in a triple transgenic mouse model of Alzheimer's disease

    doi: 10.1186/1742-2094-9-99

    Figure Lengend Snippet: Tumor necrosis factor-α gene and protein expression in the 3 × Tg mouse. (A) Fold-change (2^(− δ δCt)) is the normalized gene expression (2^(−δ Ct)) in the test sample (3 × Tg treated with Thal or 3,6′-DT) divided by the normalized gene expression (2^(−δ Ct)) in the control sample (vehicle-treated 3 × Tg). Fold-change values of less than one indicate a negative- or downregulation. Both Thal and 3,6′-DT downregulated TNFα gene expression but the value was significant only in the 3,6′-DT group ( P = 0.033). (B) TNFα protein levels are doubled in the cortex of 3 × Tg mice compared with Non-Tg mice. 3,6′-DT, but not Thal, reduced TNFα protein levels near to Non-Tg levels in 3 × Tg mice. n = 5 to 8 per group. One-way analysis of variance ( P = 0.062, * P

    Article Snippet: Enzyme-linked immunosorbent assay The levels of TNFα in culture media or mouse cortical or spleen supernatants were measured using a commercially available ELISA kit for mouse TNFα (BioLegend ELISA MAX; BioLegend, San Diego, CA, USA) according to manufacturer’s instructions.

    Techniques: Expressing, Mouse Assay

    Thalidomide and 3,6 ′ -dithiothalidomide attenuate lipopolysaccharide-induced increase in tumor necrosis factor-α. BV2 cells were treated with 1 ng/mL LPS ± Thal or 3,6′-DT for 24 h. Initial studies in BV2 cells demonstrate that both Thal and 3,6′-DT are effective at attenuating LPS-induced TNFα release into culture media. 3,6′-DT has an IC 50 value of approximately 1 μM whereas the IC 50 for Thal is > 10 μM. n = 6 per group. One-way analysis of variance: P

    Journal: Journal of Neuroinflammation

    Article Title: Early intervention with a small molecule inhibitor for tumor nefosis factor-? prevents cognitive deficits in a triple transgenic mouse model of Alzheimer's disease

    doi: 10.1186/1742-2094-9-99

    Figure Lengend Snippet: Thalidomide and 3,6 ′ -dithiothalidomide attenuate lipopolysaccharide-induced increase in tumor necrosis factor-α. BV2 cells were treated with 1 ng/mL LPS ± Thal or 3,6′-DT for 24 h. Initial studies in BV2 cells demonstrate that both Thal and 3,6′-DT are effective at attenuating LPS-induced TNFα release into culture media. 3,6′-DT has an IC 50 value of approximately 1 μM whereas the IC 50 for Thal is > 10 μM. n = 6 per group. One-way analysis of variance: P

    Article Snippet: Enzyme-linked immunosorbent assay The levels of TNFα in culture media or mouse cortical or spleen supernatants were measured using a commercially available ELISA kit for mouse TNFα (BioLegend ELISA MAX; BioLegend, San Diego, CA, USA) according to manufacturer’s instructions.

    Techniques:

    Tumor necrosis factor-α protein from splenocytes isolated from Non-Tg and 3 × Tg mice. Splenocytes isolated from Non-Tg and 3 × Tg mice were cultured for 24 hours and TNFα levels measured by ELISA. 3,6′-DT-treated 3 × Tg mice had significantly reduced TNFα secretion compared with vehicle-treated 3 × Tg mice. One-way analysis of variance: P = 0.0184. * P

    Journal: Journal of Neuroinflammation

    Article Title: Early intervention with a small molecule inhibitor for tumor nefosis factor-? prevents cognitive deficits in a triple transgenic mouse model of Alzheimer's disease

    doi: 10.1186/1742-2094-9-99

    Figure Lengend Snippet: Tumor necrosis factor-α protein from splenocytes isolated from Non-Tg and 3 × Tg mice. Splenocytes isolated from Non-Tg and 3 × Tg mice were cultured for 24 hours and TNFα levels measured by ELISA. 3,6′-DT-treated 3 × Tg mice had significantly reduced TNFα secretion compared with vehicle-treated 3 × Tg mice. One-way analysis of variance: P = 0.0184. * P

    Article Snippet: Enzyme-linked immunosorbent assay The levels of TNFα in culture media or mouse cortical or spleen supernatants were measured using a commercially available ELISA kit for mouse TNFα (BioLegend ELISA MAX; BioLegend, San Diego, CA, USA) according to manufacturer’s instructions.

    Techniques: Isolation, Mouse Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    3,6 ′ -dithiothalidomide reduces tumor necrosis factor-α in central nervous system-infiltrating myelomonocytic/granulocytic leukocytes. (A) Non-Tg mice; (B) 3 × Tg mice; (C) 3,6′-DT mice. CNS-infiltrating leukocytes from whole mouse brains (n = 3 to 4 per group) were isolated and evaluated for the presence of CD45 hi and myelomonocytes/granulocytes (CD45 hi /Gr1 + /Ly6G hi ) by cell surface staining and flow cytometric analyses. There was a trend towards an increased percentage of CD45 hi cells and CD45 hi /Gr1 + /Ly6G hi (not significant) in 3 × Tg mice (B) relative to Non-Tg (A) mice. 3,6′-DT (C) did not alter the percentages of these cell populations. TNFα expression in the total CD45 hi population and in the granulocyte population was increased in 3 × Tg mice relative to Non-Tg mice. 3,6′-DT treatment did not reduce TNFα expression in the total CD45 hi population but specifically reduced TNFα expression in the CD45 hi /Gr1 + /Ly6G hi population ( P = 0.031). Flow cytometry results were quantified and are presented in Table 1 .

    Journal: Journal of Neuroinflammation

    Article Title: Early intervention with a small molecule inhibitor for tumor nefosis factor-? prevents cognitive deficits in a triple transgenic mouse model of Alzheimer's disease

    doi: 10.1186/1742-2094-9-99

    Figure Lengend Snippet: 3,6 ′ -dithiothalidomide reduces tumor necrosis factor-α in central nervous system-infiltrating myelomonocytic/granulocytic leukocytes. (A) Non-Tg mice; (B) 3 × Tg mice; (C) 3,6′-DT mice. CNS-infiltrating leukocytes from whole mouse brains (n = 3 to 4 per group) were isolated and evaluated for the presence of CD45 hi and myelomonocytes/granulocytes (CD45 hi /Gr1 + /Ly6G hi ) by cell surface staining and flow cytometric analyses. There was a trend towards an increased percentage of CD45 hi cells and CD45 hi /Gr1 + /Ly6G hi (not significant) in 3 × Tg mice (B) relative to Non-Tg (A) mice. 3,6′-DT (C) did not alter the percentages of these cell populations. TNFα expression in the total CD45 hi population and in the granulocyte population was increased in 3 × Tg mice relative to Non-Tg mice. 3,6′-DT treatment did not reduce TNFα expression in the total CD45 hi population but specifically reduced TNFα expression in the CD45 hi /Gr1 + /Ly6G hi population ( P = 0.031). Flow cytometry results were quantified and are presented in Table 1 .

    Article Snippet: Enzyme-linked immunosorbent assay The levels of TNFα in culture media or mouse cortical or spleen supernatants were measured using a commercially available ELISA kit for mouse TNFα (BioLegend ELISA MAX; BioLegend, San Diego, CA, USA) according to manufacturer’s instructions.

    Techniques: Mouse Assay, Isolation, Staining, Flow Cytometry, Expressing, Cytometry

    Genetic deficiency of RIP3 protects from TNFα-mediated shock in the presence and absence of caspase inhibition. Wild-type mice (open circles) and RIP3-deficient mice (black squares, n = 8 per group) were treated with mouse TNFα in the absence (A) or presence (B) of zVAD. Survival is presented as a Kaplan-Meyer plot. Genetic RIP3-deficiency protects from both TNFα-mediated shock ( P

    Journal: Molecular Medicine

    Article Title: Dichotomy between RIP1- and RIP3-Mediated Necroptosis in Tumor Necrosis Factor-?-Induced Shock

    doi: 10.2119/molmed.2011.00423

    Figure Lengend Snippet: Genetic deficiency of RIP3 protects from TNFα-mediated shock in the presence and absence of caspase inhibition. Wild-type mice (open circles) and RIP3-deficient mice (black squares, n = 8 per group) were treated with mouse TNFα in the absence (A) or presence (B) of zVAD. Survival is presented as a Kaplan-Meyer plot. Genetic RIP3-deficiency protects from both TNFα-mediated shock ( P

    Article Snippet: Recombinant mouse TNFα (carrier-free) and purified α-human TNFα were purchased from BioLegend (Uithoorn, Netherlands).

    Techniques: Inhibition, Mouse Assay

    TAT-crmA inhibits both apoptosis and necroptosis in vitro . (A) Jurkat T cells were left untreated or treated with recombinant human TNFα plus CHX, zVAD and TAT-crmA as indicated. After 5 h of incubation at 37°C, cells were stained for phosphatidylserine exposure with annexin V-FITC antibody and analyzed by FACS. Presence of zVAD or TAT-crmA prevented TNFα/CHX-induced apoptotic cell death. (B) L929, HT29 and FADD −/− Jurkat cells were left untreated or treated with TNFα plus CHX, zVAD, Nec-1 and TAT-crmA as indicated. After 5 h of incubation at 37°C, cells were stained with annexin V-FITC and analyzed by FACS. Necroptosis was induced by TNFα/CHX + zVAD and protection from necroptotic cell death was achieved by the addition of Nec-1 or TAT-crmA. (C) Western blot of cyclophilin A in the supernatant of L929 cells that were left untreated or treated for 7 h with TNFα, zVAD, Nec-1, TAT-crmA or mut.TAT-crmA, as indicated.

    Journal: Molecular Medicine

    Article Title: Dichotomy between RIP1- and RIP3-Mediated Necroptosis in Tumor Necrosis Factor-?-Induced Shock

    doi: 10.2119/molmed.2011.00423

    Figure Lengend Snippet: TAT-crmA inhibits both apoptosis and necroptosis in vitro . (A) Jurkat T cells were left untreated or treated with recombinant human TNFα plus CHX, zVAD and TAT-crmA as indicated. After 5 h of incubation at 37°C, cells were stained for phosphatidylserine exposure with annexin V-FITC antibody and analyzed by FACS. Presence of zVAD or TAT-crmA prevented TNFα/CHX-induced apoptotic cell death. (B) L929, HT29 and FADD −/− Jurkat cells were left untreated or treated with TNFα plus CHX, zVAD, Nec-1 and TAT-crmA as indicated. After 5 h of incubation at 37°C, cells were stained with annexin V-FITC and analyzed by FACS. Necroptosis was induced by TNFα/CHX + zVAD and protection from necroptotic cell death was achieved by the addition of Nec-1 or TAT-crmA. (C) Western blot of cyclophilin A in the supernatant of L929 cells that were left untreated or treated for 7 h with TNFα, zVAD, Nec-1, TAT-crmA or mut.TAT-crmA, as indicated.

    Article Snippet: Recombinant mouse TNFα (carrier-free) and purified α-human TNFα were purchased from BioLegend (Uithoorn, Netherlands).

    Techniques: In Vitro, Recombinant, Incubation, Staining, FACS, Western Blot

    Blocking necroptosis accelerates death and worsens organ damage after TNFα/zVAD-mediated hyperacute shock. (A) Mice were left untreated (open circles), injected intravenously with mouse TNFα alone (black squares) or in addition with zVAD (open triangles), zVAD + Nec-1 (open squares) or zVAD + TAT-crmA (black circles). Survival is presented as a Kaplan-Meyer plot. Whereas TNFα-treated mice die significantly later than TNFα/zVAD-treated animals ( P

    Journal: Molecular Medicine

    Article Title: Dichotomy between RIP1- and RIP3-Mediated Necroptosis in Tumor Necrosis Factor-?-Induced Shock

    doi: 10.2119/molmed.2011.00423

    Figure Lengend Snippet: Blocking necroptosis accelerates death and worsens organ damage after TNFα/zVAD-mediated hyperacute shock. (A) Mice were left untreated (open circles), injected intravenously with mouse TNFα alone (black squares) or in addition with zVAD (open triangles), zVAD + Nec-1 (open squares) or zVAD + TAT-crmA (black circles). Survival is presented as a Kaplan-Meyer plot. Whereas TNFα-treated mice die significantly later than TNFα/zVAD-treated animals ( P

    Article Snippet: Recombinant mouse TNFα (carrier-free) and purified α-human TNFα were purchased from BioLegend (Uithoorn, Netherlands).

    Techniques: Blocking Assay, Mouse Assay, Injection

    Effects of MI-2 on inflammatory TNFα production in macrophages. ( a ) TNFα concentrations in the culture supernatants from untreated or LPS-treated macrophages, with or without MI-2 treatment, as measured by ELISA. Pooled results are shown from 3 independent experiments. ** and *** indicate significant differences based on 2-tailed unpaired Student’s t -tests at P

    Journal: Scientific Reports

    Article Title: Mucosa-associated lymphoid tissue lymphoma translocation 1 as a novel therapeutic target for rheumatoid arthritis

    doi: 10.1038/s41598-017-12349-9

    Figure Lengend Snippet: Effects of MI-2 on inflammatory TNFα production in macrophages. ( a ) TNFα concentrations in the culture supernatants from untreated or LPS-treated macrophages, with or without MI-2 treatment, as measured by ELISA. Pooled results are shown from 3 independent experiments. ** and *** indicate significant differences based on 2-tailed unpaired Student’s t -tests at P

    Article Snippet: The culture supernatants were collected and kept at −80 °C until analysis, and TNFα levels in the culture supernatants were measured using the Human TNFα ELISA MAXTM Deluxe Kit following the manufacturer’s procedure (BioLegend).

    Techniques: Enzyme-linked Immunosorbent Assay