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  • 97
    Millipore gm csf
    Schematic of the dual-sized microparticle (dMP) system. The dMP formulation is an injectable platform that provides sustained extracellular release of a DC chemokine, <t>GM-CSF,</t> and a protolerogenic factor, TGF-β1, via ∼30 μm <t>nonphagocytosable</t> MPs to recruit and condition DCs at a subcutaneous injection site. Concurrently, ∼1 μm phagocytosable MPs encapsulating antigen, denatured insulin, and a tolerizing agent, vitamin D 3 , provide targeted intracellular delivery to the locally recruited DCs in order to promote presentation of the T1D autoantigen in a tolerogenic context.
    Gm Csf, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1675 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    R&D Systems granulocyte macrophage csf gm csf
    Schematic of the dual-sized microparticle (dMP) system. The dMP formulation is an injectable platform that provides sustained extracellular release of a DC chemokine, <t>GM-CSF,</t> and a protolerogenic factor, TGF-β1, via ∼30 μm <t>nonphagocytosable</t> MPs to recruit and condition DCs at a subcutaneous injection site. Concurrently, ∼1 μm phagocytosable MPs encapsulating antigen, denatured insulin, and a tolerizing agent, vitamin D 3 , provide targeted intracellular delivery to the locally recruited DCs in order to promote presentation of the T1D autoantigen in a tolerogenic context.
    Granulocyte Macrophage Csf Gm Csf, supplied by R&D Systems, used in various techniques. Bioz Stars score: 88/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore rat gm csf
    Schematic of the dual-sized microparticle (dMP) system. The dMP formulation is an injectable platform that provides sustained extracellular release of a DC chemokine, <t>GM-CSF,</t> and a protolerogenic factor, TGF-β1, via ∼30 μm <t>nonphagocytosable</t> MPs to recruit and condition DCs at a subcutaneous injection site. Concurrently, ∼1 μm phagocytosable MPs encapsulating antigen, denatured insulin, and a tolerizing agent, vitamin D 3 , provide targeted intracellular delivery to the locally recruited DCs in order to promote presentation of the T1D autoantigen in a tolerogenic context.
    Rat Gm Csf, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Amgen granulocyte macrophage colony stimulating factor
    DCs loaded with killed breast cancer cells induce differentiation of naïve CD8 + T cells into effector cells. Sorted naïve CD8 + T were cultured for 3 weeks with autologous DCs either (a) not loaded or (b) loaded with killed breast cancer cells. The cultures were restimulated twice. Intracytoplasmic staining and flow cytometry are given for the indicated effector molecules. Shown is one representative experiment from three with T cells from three different healthy donors. Percentage of CD8 + T cells expressing (c) indicated cytokines or (d) effector molecules in cultures with unloaded (black bars) and loaded (gray bars) DCs. DC, dendritic cell; GM-CSF, <t>granulocyte-macrophage</t> <t>colony-stimulating</t> <t>factor;</t> Gr, granzyme; IFN, interferon; IL, interleukin.
    Granulocyte Macrophage Colony Stimulating Factor, supplied by Amgen, used in various techniques. Bioz Stars score: 92/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Amoytop Biotech gm csf
    DCs loaded with killed breast cancer cells induce differentiation of naïve CD8 + T cells into effector cells. Sorted naïve CD8 + T were cultured for 3 weeks with autologous DCs either (a) not loaded or (b) loaded with killed breast cancer cells. The cultures were restimulated twice. Intracytoplasmic staining and flow cytometry are given for the indicated effector molecules. Shown is one representative experiment from three with T cells from three different healthy donors. Percentage of CD8 + T cells expressing (c) indicated cytokines or (d) effector molecules in cultures with unloaded (black bars) and loaded (gray bars) DCs. DC, dendritic cell; GM-CSF, <t>granulocyte-macrophage</t> <t>colony-stimulating</t> <t>factor;</t> Gr, granzyme; IFN, interferon; IL, interleukin.
    Gm Csf, supplied by Amoytop Biotech, used in various techniques. Bioz Stars score: 93/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson gm csf
    Cytokine secretion by dNK cells in response to KIR2DS4 activation. ( A and B ) A semiquantitative fluorescent chip-based sandwich ELISA was used to screen for 120 cytokines in supernatants taken from mixed decidual leukocytes of KIR2DS4 + donors (see Supplemental Table I ). Leukocytes were cultured on Ab-coated plastic for 12–24 h, where the only cells to express KIR2DS4 were the dNK cells. Fluorescent spots for cytokines of interest are highlighted in (A). The cropped regions of interest are taken from different chips and different donors. They are grouped according to whether they show a > 1.5-fold increase in secretion on average across all donors (Increase); secretion that was already high within the isotype control stimulation, so the screen was insensitive (Ambiguous); and control spots (Control). (B) The cytokines that were upregulated > 1.25-fold upon KIR2DS4 activation in at least one of four donors tested are listed in the table. The mean fold change across all four donors is shown to the right. Values > 1.25-fold are highlighted in gray. ( C – E ) Mixed decidual leukocytes were cultured on plastic coated with either anti-KIR2DS4 Ab or an isotype control (IgG2a) in the presence of monensin and brefeldin A. After 5 h, cells were fixed and live CD56 + CD9 + KIR2DS4 + dNK cells were identified by flow cytometry (C). Although KIR2DS4 expression reduced upon cross-linking (C), most retained KIR2DS4 expression. These KIR2DS4 + dNK cells were assessed for intracellular cytokines: (D) <t>GM-CSF</t> ( n = 7) and (E) <t>CCL3</t> ( n = 7). ( F ) When Abs for flow cytometry were not available, purified dNK cells were cultured on Ab-coated plastic for 12–48 h and the production of CCL1 and XCL1 ( n = 8) was detected in supernatants by commercial sandwich ELISA. Results are color coded according to donor. * p
    Gm Csf, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 1344 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    BioLegend gm csf
    <t>GM-CSF</t> production by human T cells. (A) Representative flow cytometric analysis results for GM-CSF and <t>IFN-γ</t> production by unstimulated human peripheral blood CD4 + and CD4 − T cells, or after stimulation with M. tuberculosis lysate
    Gm Csf, supplied by BioLegend, used in various techniques. Bioz Stars score: 93/100, based on 599 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cellgenix gm csf
    Stimuli effects on individual genes A-D . Volcano plots representing the gene expression changes (x-axis: log fold) together with the statistical significance (y-axis: -10log p-value). Each condition was compared to the respective unstimulated cell type. Genes with a log fold change of more than 10 were labeled with the gene name. A) <t>GM-CSF</t> stimulated CD1c + mDC; B) pRNA stimulated CD1c + mDCs; C) <t>FSME</t> stimulated pDCs; D) pRNA stimulated pDCs.
    Gm Csf, supplied by Cellgenix, used in various techniques. Bioz Stars score: 94/100, based on 364 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Fisher Scientific gm csf
    Stimuli effects on individual genes A-D . Volcano plots representing the gene expression changes (x-axis: log fold) together with the statistical significance (y-axis: -10log p-value). Each condition was compared to the respective unstimulated cell type. Genes with a log fold change of more than 10 were labeled with the gene name. A) <t>GM-CSF</t> stimulated CD1c + mDC; B) pRNA stimulated CD1c + mDCs; C) <t>FSME</t> stimulated pDCs; D) pRNA stimulated pDCs.
    Gm Csf, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 92/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GENTAUR gm csf
    COMBIG/Ad5M-matured <t>DCs</t> express a mature phenotype and Th1-polarized cytokine secretion profile. (A) CD14 + monocytes were isolated from healthy donor PBMCs, differentiated into imDCs by <t>GM-CSF/IL-4</t> for 5 days, matured under different conditions for 18 h, washed and further cultured for 24 h and analyzed. (B) DCs were characterized for HLA-DR, CD40, CD80, CD86 and CD83 expression by flow cytometry. Mean fluorescence intensity (MFI) for each marker on DCs (CD14 − CD1a + ) produced from eight donors are shown. (C) Secreted cytokines were assessed in supernatants of each treatment by proteome profiler where supernatants from six donors were pooled. (D) IL-12, IL-6 and CXCL10 secretion were also verified by ELISA for each donor. (E, F) Inflammasome activation was evaluated by the re-localization of the protein ASC, an inflammasome component, from a diffuse state to a single speck exhibited in representative FACS plots of ASC width (ASC-W) and ASC area (ASC-A) and % of speck + DCs produced from six donors. Data are shown as mean±SEM (n.s. p ≥ 0.05; * P
    Gm Csf, supplied by GENTAUR, used in various techniques. Bioz Stars score: 92/100, based on 336 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ImmunoTools gm csf
    Monocyte-derived dendritic cells and macrophages are barely sensitive for BV6-induced cell death. (A and B) Monocyte-derived macrophages, immature and Fc-CD40L maturated monocyte-derived dendritic cells were challenged for one day with and without 10 µM BV6. Cells were then analyzed by annexin-V staining for cell death induction (A; upper panel: representative analysis of one individual sample; lower panel: summary of the data of 3 independent DC experiments and 6 independent experiments with monocytes and macrophages). p100 processing were determined by western blotting and is shown for one representative experiment (B). (C) Monocytes cultivated overnight in <t>GM-CSF/IL4,</t> iDCs obtained after 7 days of cultivation with GM-CSF/IL4 and mDCs maturated with TNF or Fc-CD40L were analyzed by western blotting with respect to the expression of the indicated proteins (data shown are representative for four independent experiments).
    Gm Csf, supplied by ImmunoTools, used in various techniques. Bioz Stars score: 95/100, based on 553 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    MedImmune gm csf
    Monocyte-derived dendritic cells and macrophages are barely sensitive for BV6-induced cell death. (A and B) Monocyte-derived macrophages, immature and Fc-CD40L maturated monocyte-derived dendritic cells were challenged for one day with and without 10 µM BV6. Cells were then analyzed by annexin-V staining for cell death induction (A; upper panel: representative analysis of one individual sample; lower panel: summary of the data of 3 independent DC experiments and 6 independent experiments with monocytes and macrophages). p100 processing were determined by western blotting and is shown for one representative experiment (B). (C) Monocytes cultivated overnight in <t>GM-CSF/IL4,</t> iDCs obtained after 7 days of cultivation with GM-CSF/IL4 and mDCs maturated with TNF or Fc-CD40L were analyzed by western blotting with respect to the expression of the indicated proteins (data shown are representative for four independent experiments).
    Gm Csf, supplied by MedImmune, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Novartis gm csf
    Effect of internalized p41 fragment on the proteolytic activity of cysteine proteases (A) and the secretion of IL-12/p70 (B, C). Samples (A): cell lysates of non-treated immature DC, immature DC after a 6-h incubation with p41 fragment (0.035 μM, 0.35 μM and 3.5 μM) and non-treated immature DC with 10 μM E-64. Fluorogenic substrate in buffer with DTT was used as blank (BLK). Representative measurements, each of three biological repetitions, are shown. Samples (B and C): cell free supernatants (culture media) of immature DC, preincubated with p41 fragment (0.035 μM, 0.35 μM and 3.5 μM) for 6 h prior to their maturation with TNF-α (B) or <t>LPS</t> (C). Non-treated cells (no preincubation with p41 fragment): immature DC, cultured in the presence of <t>GM-CSF</t> for three days (no maturation), DC matured with TNF-α, and DC matured with LPS. Pretreated but non-matured cells: immature DC, pretreated with 3.5 μM p41 fragment, and cultured in the presence of GM-CSF. IL-12 concentrations (in pg/ml) were measured in triplicate, average values ± SD are shown. (D) Immunolabelled cathepsins L and S in DC lysates. Samples (50 μg per well): (1) immature DC, (2) DC pretreated with 3.5 μM p41 fragment for 6 h, no LPS, (3) DC matured with LPS for 24 h, (4) DC pretreated with 3.5 μM p41 fragment for 6 h and matured with LPS for 24 h.
    Gm Csf, supplied by Novartis, used in various techniques. Bioz Stars score: 92/100, based on 212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Pharmingen gm csf
    <t>IFN-γ</t> and <t>GM-CSF</t> production of IL-15- and/or IL-18-stimulated NK cells and effects of commercial and S. griseus 10/ppi valinomycins on cytokine production. The stimulatory effects of IL-15 and IL-18 on cytokine production are synergistic. Valinomycin almost completely inhibits IL-15-induced cytokine production, whereas there is some cytokine production left in cells activated by a combination of IL-15 and IL-18 and pretreated with valinomycin. The P values in paired t tests for the differences between IFN-γ production by lymphocytes treated with IL-15 with valinomycin and that by lymphocytes treated with IL-15 and IL-18 with valinomycin are
    Gm Csf, supplied by Pharmingen, used in various techniques. Bioz Stars score: 92/100, based on 184 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Prospec gm csf
    The effect of cytokines on the development of CR3 + and CR4 + macrophages and in MDM. ( a ) Monocytes were treated with 40 ng/ml LT-α, <t>IFN-γ,</t> IL-4, IL-13, IL-1β, IL-6, IL-10, <t>M-CSF,</t> GM-CSF or dexamethasone, 20 ng/ml TNF, 15 ng/ml TGF-β1. ( b ) Monocytes were cultured for 3 days for maturation into macrophages. The MDM were then incubated for 24 h with 40 ng/ml of the cytokines, LT-α, IFN-γ, IL-4, IL-1β, IL-6, IL-10, IL-13, M-CSF, GM-CSF, 20 ng/ml TNF, 15 ng/ml TGF-β1 or Dexamethasone (50 ng/ml). The level of CD11b and CD11c mRNA was measured using qPCR. Data are expressed as fold-change over GAPDH-normalized CD11b and CD11c mRNA in the absence of cytokine set as 1. Data are presented as means ± SD of three experiments, each conducted with cells from different individuals, *p
    Gm Csf, supplied by Prospec, used in various techniques. Bioz Stars score: 94/100, based on 170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche gm csf
    The EphA2 receptor tyrosine kinase is expressed by in vitro generated LLDC Adherent mononuclear cells were isolated and cultured one week in the presence of <t>GM-CSF</t> and IL-4 (for monocyte derived dendritic cells, DC) and <t>TGF-β</t> (for monocyte derived LLDC). (A) Cells were incubated with a monoclonal EphA2 antibody (bold line) or an irrelevant antibody (dotted line) followed by staining with PE-labeled α-mouse antibody. (B) Cells were incubated with a soluble EphA2 ligand fusion protein (ephrin-A4-Fc, bold line) and a negative control fusion protein (CD19-Fc, dotted line). (C) A Western blot with cell lysates from monocyte derived dendritic cells (DC) or LLDC (LLDC) were immunoblotted using a polyclonal anti-EphA2 antiserum.
    Gm Csf, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 132 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Sanofi gm csf
    Profiling of antibody responses in cancer patients to <t>CTLA-4</t> blockade and <t>GM-CSF</t> with protein microarrays
    Gm Csf, supplied by Sanofi, used in various techniques. Bioz Stars score: 92/100, based on 124 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Schering-Plough gm csf
    Tumor environmental factors turn monocytes into CD163 high CD86 low IL‐10 high MΦ. HD CD14 + monocytes were cultured in the presence of 25% SNDils for 7 days, and surface markers and cytokine production were evaluated 24 h after addition of LPS. (a) r‐CD163 MFI from control APCs ( n = 8 independent donors) and SNDil‐MΦ ( n = 29 independent SNDils). (b) Representative pseudocolour plots of CD64 + CD163 + cells of <t>M0‐MΦ</t> and SNDil‐MΦ based on the median obtained within each group. (c) Expression of CD86 and (d) production of TNF‐α and IL‐10 in CD163 low and CD163 high SNDil‐MΦ or in control APCs. For (a–d) , experiments performed with, at least, five independent donor monocytes and different SNDils ( n = 29). (e) Correlation of r‐CD163 MFI and IL‐10 production among SNDil‐MΦ. (f, g) SNDil‐MΦ were cultured in the presence of neutralising <t>anti‐M‐CSF,</t> anti‐TGF‐β and anti‐VEGF or control antibodies during the differentiation process and were activated by LPS during the last 24 h to evaluate surface markers (f) and cytokine production (g) in CD163 high IL‐10 high SNDil‐MΦ (red dots, n = 4; * P ≤ 0.05) and CD163 low IL‐10 low SNDil‐MΦ (blue dots; n = 3).
    Gm Csf, supplied by Schering-Plough, used in various techniques. Bioz Stars score: 92/100, based on 353 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Shenandoah Biotechnology gm csf
    Tumor environmental factors turn monocytes into CD163 high CD86 low IL‐10 high MΦ. HD CD14 + monocytes were cultured in the presence of 25% SNDils for 7 days, and surface markers and cytokine production were evaluated 24 h after addition of LPS. (a) r‐CD163 MFI from control APCs ( n = 8 independent donors) and SNDil‐MΦ ( n = 29 independent SNDils). (b) Representative pseudocolour plots of CD64 + CD163 + cells of <t>M0‐MΦ</t> and SNDil‐MΦ based on the median obtained within each group. (c) Expression of CD86 and (d) production of TNF‐α and IL‐10 in CD163 low and CD163 high SNDil‐MΦ or in control APCs. For (a–d) , experiments performed with, at least, five independent donor monocytes and different SNDils ( n = 29). (e) Correlation of r‐CD163 MFI and IL‐10 production among SNDil‐MΦ. (f, g) SNDil‐MΦ were cultured in the presence of neutralising <t>anti‐M‐CSF,</t> anti‐TGF‐β and anti‐VEGF or control antibodies during the differentiation process and were activated by LPS during the last 24 h to evaluate surface markers (f) and cytokine production (g) in CD163 high IL‐10 high SNDil‐MΦ (red dots, n = 4; * P ≤ 0.05) and CD163 low IL‐10 low SNDil‐MΦ (blue dots; n = 3).
    Gm Csf, supplied by Shenandoah Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    US Biological Life Sciences gm csf
    Tumor environmental factors turn monocytes into CD163 high CD86 low IL‐10 high MΦ. HD CD14 + monocytes were cultured in the presence of 25% SNDils for 7 days, and surface markers and cytokine production were evaluated 24 h after addition of LPS. (a) r‐CD163 MFI from control APCs ( n = 8 independent donors) and SNDil‐MΦ ( n = 29 independent SNDils). (b) Representative pseudocolour plots of CD64 + CD163 + cells of <t>M0‐MΦ</t> and SNDil‐MΦ based on the median obtained within each group. (c) Expression of CD86 and (d) production of TNF‐α and IL‐10 in CD163 low and CD163 high SNDil‐MΦ or in control APCs. For (a–d) , experiments performed with, at least, five independent donor monocytes and different SNDils ( n = 29). (e) Correlation of r‐CD163 MFI and IL‐10 production among SNDil‐MΦ. (f, g) SNDil‐MΦ were cultured in the presence of neutralising <t>anti‐M‐CSF,</t> anti‐TGF‐β and anti‐VEGF or control antibodies during the differentiation process and were activated by LPS during the last 24 h to evaluate surface markers (f) and cytokine production (g) in CD163 high IL‐10 high SNDil‐MΦ (red dots, n = 4; * P ≤ 0.05) and CD163 low IL‐10 low SNDil‐MΦ (blue dots; n = 3).
    Gm Csf, supplied by US Biological Life Sciences, used in various techniques. Bioz Stars score: 92/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Berlex Laboratories Inc gm csf
    Tumor environmental factors turn monocytes into CD163 high CD86 low IL‐10 high MΦ. HD CD14 + monocytes were cultured in the presence of 25% SNDils for 7 days, and surface markers and cytokine production were evaluated 24 h after addition of LPS. (a) r‐CD163 MFI from control APCs ( n = 8 independent donors) and SNDil‐MΦ ( n = 29 independent SNDils). (b) Representative pseudocolour plots of CD64 + CD163 + cells of <t>M0‐MΦ</t> and SNDil‐MΦ based on the median obtained within each group. (c) Expression of CD86 and (d) production of TNF‐α and IL‐10 in CD163 low and CD163 high SNDil‐MΦ or in control APCs. For (a–d) , experiments performed with, at least, five independent donor monocytes and different SNDils ( n = 29). (e) Correlation of r‐CD163 MFI and IL‐10 production among SNDil‐MΦ. (f, g) SNDil‐MΦ were cultured in the presence of neutralising <t>anti‐M‐CSF,</t> anti‐TGF‐β and anti‐VEGF or control antibodies during the differentiation process and were activated by LPS during the last 24 h to evaluate surface markers (f) and cytokine production (g) in CD163 high IL‐10 high SNDil‐MΦ (red dots, n = 4; * P ≤ 0.05) and CD163 low IL‐10 low SNDil‐MΦ (blue dots; n = 3).
    Gm Csf, supplied by Berlex Laboratories Inc, 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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    Boehringer Mannheim gm csf
    Tumor environmental factors turn monocytes into CD163 high CD86 low IL‐10 high MΦ. HD CD14 + monocytes were cultured in the presence of 25% SNDils for 7 days, and surface markers and cytokine production were evaluated 24 h after addition of LPS. (a) r‐CD163 MFI from control APCs ( n = 8 independent donors) and SNDil‐MΦ ( n = 29 independent SNDils). (b) Representative pseudocolour plots of CD64 + CD163 + cells of <t>M0‐MΦ</t> and SNDil‐MΦ based on the median obtained within each group. (c) Expression of CD86 and (d) production of TNF‐α and IL‐10 in CD163 low and CD163 high SNDil‐MΦ or in control APCs. For (a–d) , experiments performed with, at least, five independent donor monocytes and different SNDils ( n = 29). (e) Correlation of r‐CD163 MFI and IL‐10 production among SNDil‐MΦ. (f, g) SNDil‐MΦ were cultured in the presence of neutralising <t>anti‐M‐CSF,</t> anti‐TGF‐β and anti‐VEGF or control antibodies during the differentiation process and were activated by LPS during the last 24 h to evaluate surface markers (f) and cytokine production (g) in CD163 high IL‐10 high SNDil‐MΦ (red dots, n = 4; * P ≤ 0.05) and CD163 low IL‐10 low SNDil‐MΦ (blue dots; n = 3).
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    Cytogen gm csf
    Tumor environmental factors turn monocytes into CD163 high CD86 low IL‐10 high MΦ. HD CD14 + monocytes were cultured in the presence of 25% SNDils for 7 days, and surface markers and cytokine production were evaluated 24 h after addition of LPS. (a) r‐CD163 MFI from control APCs ( n = 8 independent donors) and SNDil‐MΦ ( n = 29 independent SNDils). (b) Representative pseudocolour plots of CD64 + CD163 + cells of <t>M0‐MΦ</t> and SNDil‐MΦ based on the median obtained within each group. (c) Expression of CD86 and (d) production of TNF‐α and IL‐10 in CD163 low and CD163 high SNDil‐MΦ or in control APCs. For (a–d) , experiments performed with, at least, five independent donor monocytes and different SNDils ( n = 29). (e) Correlation of r‐CD163 MFI and IL‐10 production among SNDil‐MΦ. (f, g) SNDil‐MΦ were cultured in the presence of neutralising <t>anti‐M‐CSF,</t> anti‐TGF‐β and anti‐VEGF or control antibodies during the differentiation process and were activated by LPS during the last 24 h to evaluate surface markers (f) and cytokine production (g) in CD163 high IL‐10 high SNDil‐MΦ (red dots, n = 4; * P ≤ 0.05) and CD163 low IL‐10 low SNDil‐MΦ (blue dots; n = 3).
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    Tumor environmental factors turn monocytes into CD163 high CD86 low IL‐10 high MΦ. HD CD14 + monocytes were cultured in the presence of 25% SNDils for 7 days, and surface markers and cytokine production were evaluated 24 h after addition of LPS. (a) r‐CD163 MFI from control APCs ( n = 8 independent donors) and SNDil‐MΦ ( n = 29 independent SNDils). (b) Representative pseudocolour plots of CD64 + CD163 + cells of <t>M0‐MΦ</t> and SNDil‐MΦ based on the median obtained within each group. (c) Expression of CD86 and (d) production of TNF‐α and IL‐10 in CD163 low and CD163 high SNDil‐MΦ or in control APCs. For (a–d) , experiments performed with, at least, five independent donor monocytes and different SNDils ( n = 29). (e) Correlation of r‐CD163 MFI and IL‐10 production among SNDil‐MΦ. (f, g) SNDil‐MΦ were cultured in the presence of neutralising <t>anti‐M‐CSF,</t> anti‐TGF‐β and anti‐VEGF or control antibodies during the differentiation process and were activated by LPS during the last 24 h to evaluate surface markers (f) and cytokine production (g) in CD163 high IL‐10 high SNDil‐MΦ (red dots, n = 4; * P ≤ 0.05) and CD163 low IL‐10 low SNDil‐MΦ (blue dots; n = 3).
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    Monocyte genes induced by both <t>GM-CSF</t> and 15 <t>kDa</t> granulysin . Hierarchical clustering of the 3191, 2416, 1534 and 1738 genes induced by both factors at 4 (a), 12 (b), 24 (c) and 48 (d) hours respectively (p-value
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    Image Search Results


    Schematic of the dual-sized microparticle (dMP) system. The dMP formulation is an injectable platform that provides sustained extracellular release of a DC chemokine, GM-CSF, and a protolerogenic factor, TGF-β1, via ∼30 μm nonphagocytosable MPs to recruit and condition DCs at a subcutaneous injection site. Concurrently, ∼1 μm phagocytosable MPs encapsulating antigen, denatured insulin, and a tolerizing agent, vitamin D 3 , provide targeted intracellular delivery to the locally recruited DCs in order to promote presentation of the T1D autoantigen in a tolerogenic context.

    Journal: ACS Biomaterials Science & Engineering

    Article Title: Dual-Sized Microparticle System for Generating Suppressive Dendritic Cells Prevents and Reverses Type 1 Diabetes in the Nonobese Diabetic Mouse Model

    doi: 10.1021/acsbiomaterials.9b00332

    Figure Lengend Snippet: Schematic of the dual-sized microparticle (dMP) system. The dMP formulation is an injectable platform that provides sustained extracellular release of a DC chemokine, GM-CSF, and a protolerogenic factor, TGF-β1, via ∼30 μm nonphagocytosable MPs to recruit and condition DCs at a subcutaneous injection site. Concurrently, ∼1 μm phagocytosable MPs encapsulating antigen, denatured insulin, and a tolerizing agent, vitamin D 3 , provide targeted intracellular delivery to the locally recruited DCs in order to promote presentation of the T1D autoantigen in a tolerogenic context.

    Article Snippet: Briefly, phagocytosable MPs were loaded with 1-α,25-dihydroxyvitamin D3 (VD3; Thermo Fisher Scientific, Waltham, MA) or denatured human recombinant insulin (Sigma-Aldrich, St. Louis, MO) while nonphagocytosable MPs encapsulated TGF-β1 or GM-CSF (Millipore Sigma).

    Techniques: Injection

    dMP administration prevents diabetes onset in NOD mice. A cohort of 8-week-old NOD mice ( n = 10/group) were injected at a subcutaneous site anatomically proximal to the pancreas with the described MP formulations over 16 weeks. Animals received MP injections (arrows) once a week for the first 3 weeks (8, 9, and 10 weeks of age) and a booster injection once monthly thereafter for 4 months (12, 16, 20, and 24 weeks of age). Unloaded MPs, a soluble bolus of factors without MPs, and omission of factors were investigated. When a factor-loaded MP was omitted, unloaded MPs were delivered to deliver an equivalent PLGA mass. Animals were monitored weekly until week 28, and mice were considered diabetic when blood glucose levels were ≥240 mg/dL on 2 consecutive days. The full dMP (solid line with solid tilted square, VD 3 /TGF-β1/GM-CSF/insulin MPs) and unloaded MPs groups were replotted alongside different experimental groups to highlight the requirement of MP encapsulation (A), antigen (B), and the full dMP formulation (C) in order to see maximum therapeutic effect. Survival data are fit using the Kaplan–Meier nonparametric survival analysis model, and statistical analysis was performed via log-rank test (Mantel–Cox method). Statistical significance was not realized when accounting for multiple comparisons via Bonferroni correction, as the study was not powered to resolve this large number of groups. However, pairwise comparison between survival curves of mice that received the dMP and mice that received unloaded MPs resulted in a P -value of

    Journal: ACS Biomaterials Science & Engineering

    Article Title: Dual-Sized Microparticle System for Generating Suppressive Dendritic Cells Prevents and Reverses Type 1 Diabetes in the Nonobese Diabetic Mouse Model

    doi: 10.1021/acsbiomaterials.9b00332

    Figure Lengend Snippet: dMP administration prevents diabetes onset in NOD mice. A cohort of 8-week-old NOD mice ( n = 10/group) were injected at a subcutaneous site anatomically proximal to the pancreas with the described MP formulations over 16 weeks. Animals received MP injections (arrows) once a week for the first 3 weeks (8, 9, and 10 weeks of age) and a booster injection once monthly thereafter for 4 months (12, 16, 20, and 24 weeks of age). Unloaded MPs, a soluble bolus of factors without MPs, and omission of factors were investigated. When a factor-loaded MP was omitted, unloaded MPs were delivered to deliver an equivalent PLGA mass. Animals were monitored weekly until week 28, and mice were considered diabetic when blood glucose levels were ≥240 mg/dL on 2 consecutive days. The full dMP (solid line with solid tilted square, VD 3 /TGF-β1/GM-CSF/insulin MPs) and unloaded MPs groups were replotted alongside different experimental groups to highlight the requirement of MP encapsulation (A), antigen (B), and the full dMP formulation (C) in order to see maximum therapeutic effect. Survival data are fit using the Kaplan–Meier nonparametric survival analysis model, and statistical analysis was performed via log-rank test (Mantel–Cox method). Statistical significance was not realized when accounting for multiple comparisons via Bonferroni correction, as the study was not powered to resolve this large number of groups. However, pairwise comparison between survival curves of mice that received the dMP and mice that received unloaded MPs resulted in a P -value of

    Article Snippet: Briefly, phagocytosable MPs were loaded with 1-α,25-dihydroxyvitamin D3 (VD3; Thermo Fisher Scientific, Waltham, MA) or denatured human recombinant insulin (Sigma-Aldrich, St. Louis, MO) while nonphagocytosable MPs encapsulated TGF-β1 or GM-CSF (Millipore Sigma).

    Techniques: Mouse Assay, Injection

    DCs loaded with killed breast cancer cells induce differentiation of naïve CD8 + T cells into effector cells. Sorted naïve CD8 + T were cultured for 3 weeks with autologous DCs either (a) not loaded or (b) loaded with killed breast cancer cells. The cultures were restimulated twice. Intracytoplasmic staining and flow cytometry are given for the indicated effector molecules. Shown is one representative experiment from three with T cells from three different healthy donors. Percentage of CD8 + T cells expressing (c) indicated cytokines or (d) effector molecules in cultures with unloaded (black bars) and loaded (gray bars) DCs. DC, dendritic cell; GM-CSF, granulocyte-macrophage colony-stimulating factor; Gr, granzyme; IFN, interferon; IL, interleukin.

    Journal: Breast Cancer Research

    Article Title: Cross-priming of cyclin B1, MUC-1 and survivin-specific CD8+ T cells by dendritic cells loaded with killed allogeneic breast cancer cells

    doi: 10.1186/bcr1621

    Figure Lengend Snippet: DCs loaded with killed breast cancer cells induce differentiation of naïve CD8 + T cells into effector cells. Sorted naïve CD8 + T were cultured for 3 weeks with autologous DCs either (a) not loaded or (b) loaded with killed breast cancer cells. The cultures were restimulated twice. Intracytoplasmic staining and flow cytometry are given for the indicated effector molecules. Shown is one representative experiment from three with T cells from three different healthy donors. Percentage of CD8 + T cells expressing (c) indicated cytokines or (d) effector molecules in cultures with unloaded (black bars) and loaded (gray bars) DCs. DC, dendritic cell; GM-CSF, granulocyte-macrophage colony-stimulating factor; Gr, granzyme; IFN, interferon; IL, interleukin.

    Article Snippet: Cytokines used included granulocyte-macrophage colony-stimulating factor (100 ng/ml; Immunex, Amgen Thousand Oaks, CA, USA), IL-4 (25 ng/ml; R & D Systems, Minneapolis, MN, USA), soluble CD40 ligand (200 ng/ml; R & D Systems), IL-2 (10 IU/ml; R & D Systems), and IL-7 (10 IU/ml; R & D Systems).

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

    Cytokine secretion by dNK cells in response to KIR2DS4 activation. ( A and B ) A semiquantitative fluorescent chip-based sandwich ELISA was used to screen for 120 cytokines in supernatants taken from mixed decidual leukocytes of KIR2DS4 + donors (see Supplemental Table I ). Leukocytes were cultured on Ab-coated plastic for 12–24 h, where the only cells to express KIR2DS4 were the dNK cells. Fluorescent spots for cytokines of interest are highlighted in (A). The cropped regions of interest are taken from different chips and different donors. They are grouped according to whether they show a > 1.5-fold increase in secretion on average across all donors (Increase); secretion that was already high within the isotype control stimulation, so the screen was insensitive (Ambiguous); and control spots (Control). (B) The cytokines that were upregulated > 1.25-fold upon KIR2DS4 activation in at least one of four donors tested are listed in the table. The mean fold change across all four donors is shown to the right. Values > 1.25-fold are highlighted in gray. ( C – E ) Mixed decidual leukocytes were cultured on plastic coated with either anti-KIR2DS4 Ab or an isotype control (IgG2a) in the presence of monensin and brefeldin A. After 5 h, cells were fixed and live CD56 + CD9 + KIR2DS4 + dNK cells were identified by flow cytometry (C). Although KIR2DS4 expression reduced upon cross-linking (C), most retained KIR2DS4 expression. These KIR2DS4 + dNK cells were assessed for intracellular cytokines: (D) GM-CSF ( n = 7) and (E) CCL3 ( n = 7). ( F ) When Abs for flow cytometry were not available, purified dNK cells were cultured on Ab-coated plastic for 12–48 h and the production of CCL1 and XCL1 ( n = 8) was detected in supernatants by commercial sandwich ELISA. Results are color coded according to donor. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Activating KIR2DS4 Is Expressed by Uterine NK Cells and Contributes to Successful Pregnancy

    doi: 10.4049/jimmunol.1601279

    Figure Lengend Snippet: Cytokine secretion by dNK cells in response to KIR2DS4 activation. ( A and B ) A semiquantitative fluorescent chip-based sandwich ELISA was used to screen for 120 cytokines in supernatants taken from mixed decidual leukocytes of KIR2DS4 + donors (see Supplemental Table I ). Leukocytes were cultured on Ab-coated plastic for 12–24 h, where the only cells to express KIR2DS4 were the dNK cells. Fluorescent spots for cytokines of interest are highlighted in (A). The cropped regions of interest are taken from different chips and different donors. They are grouped according to whether they show a > 1.5-fold increase in secretion on average across all donors (Increase); secretion that was already high within the isotype control stimulation, so the screen was insensitive (Ambiguous); and control spots (Control). (B) The cytokines that were upregulated > 1.25-fold upon KIR2DS4 activation in at least one of four donors tested are listed in the table. The mean fold change across all four donors is shown to the right. Values > 1.25-fold are highlighted in gray. ( C – E ) Mixed decidual leukocytes were cultured on plastic coated with either anti-KIR2DS4 Ab or an isotype control (IgG2a) in the presence of monensin and brefeldin A. After 5 h, cells were fixed and live CD56 + CD9 + KIR2DS4 + dNK cells were identified by flow cytometry (C). Although KIR2DS4 expression reduced upon cross-linking (C), most retained KIR2DS4 expression. These KIR2DS4 + dNK cells were assessed for intracellular cytokines: (D) GM-CSF ( n = 7) and (E) CCL3 ( n = 7). ( F ) When Abs for flow cytometry were not available, purified dNK cells were cultured on Ab-coated plastic for 12–48 h and the production of CCL1 and XCL1 ( n = 8) was detected in supernatants by commercial sandwich ELISA. Results are color coded according to donor. * p

    Article Snippet: Intracellular staining was performed according to the manufacturers’ instructions with Abs against Ki647 (BD Pharmingen), CCL3 (R & D Systems), and GM-CSF (BD Biosciences).

    Techniques: Activation Assay, Chromatin Immunoprecipitation, Sandwich ELISA, Cell Culture, Flow Cytometry, Cytometry, Expressing, Purification

    GM-CSF production by human T cells. (A) Representative flow cytometric analysis results for GM-CSF and IFN-γ production by unstimulated human peripheral blood CD4 + and CD4 − T cells, or after stimulation with M. tuberculosis lysate

    Journal: mBio

    Article Title: Role of Granulocyte-Macrophage Colony-Stimulating Factor Production by T Cells during Mycobacterium tuberculosis Infection

    doi: 10.1128/mBio.01514-17

    Figure Lengend Snippet: GM-CSF production by human T cells. (A) Representative flow cytometric analysis results for GM-CSF and IFN-γ production by unstimulated human peripheral blood CD4 + and CD4 − T cells, or after stimulation with M. tuberculosis lysate

    Article Snippet: Monoclonal antibodies against human IFN-γ (4S.B3) and GM-CSF (BVD2-21C11) were obtained from BioLegend.

    Techniques: Flow Cytometry

    Stimuli effects on individual genes A-D . Volcano plots representing the gene expression changes (x-axis: log fold) together with the statistical significance (y-axis: -10log p-value). Each condition was compared to the respective unstimulated cell type. Genes with a log fold change of more than 10 were labeled with the gene name. A) GM-CSF stimulated CD1c + mDC; B) pRNA stimulated CD1c + mDCs; C) FSME stimulated pDCs; D) pRNA stimulated pDCs.

    Journal: Oncotarget

    Article Title: Harnessing RNA sequencing for global, unbiased evaluation of two new adjuvants for dendritic-cell immunotherapy

    doi: 10.18632/oncotarget.15190

    Figure Lengend Snippet: Stimuli effects on individual genes A-D . Volcano plots representing the gene expression changes (x-axis: log fold) together with the statistical significance (y-axis: -10log p-value). Each condition was compared to the respective unstimulated cell type. Genes with a log fold change of more than 10 were labeled with the gene name. A) GM-CSF stimulated CD1c + mDC; B) pRNA stimulated CD1c + mDCs; C) FSME stimulated pDCs; D) pRNA stimulated pDCs.

    Article Snippet: DCs were stimulated with: FSME (5%; Baxter AG, Vienna), pRNA (15μg/ml), CpG-P (5μg/ml; Miltenyi Biotech, Germany), GM-CSF (800 U/ml; (Cellgenix, Freiburg, Germany). pRNA complexes were prepared fresh 5-10 minutes before adding to the cell culture. pDCs were cultured with 10 ng/mL IL-3 (Cellgenix, Freiburg, Germany) as a survival factor in addition to the stimuli.

    Techniques: Expressing, Labeling

    RNA-seq results represent protein levels A . Flow cytometry histograms for three maturation markers (CD80, PD-L1 and CD40) on pDCs and CD1c + mDCs. For CD1c: Light grey represents unstimulated samples, dark grey represents GM-CSF samples, and black represents pRNA samples. For pDC: bright transparent grey represents the unstimulated samples, light grey represents the IL-3 samples, grey represents FSME and black represents pRNA samples. B . Gene expression levels of the 3 donors of the RNA-seq. C . Correlation plot of the RNA-seq counts/1 million reads set out against the geometric mean fluorescence intensity as measured by flow cytometry (protein level). Throughout this paper, error bars represent standard error of the mean.

    Journal: Oncotarget

    Article Title: Harnessing RNA sequencing for global, unbiased evaluation of two new adjuvants for dendritic-cell immunotherapy

    doi: 10.18632/oncotarget.15190

    Figure Lengend Snippet: RNA-seq results represent protein levels A . Flow cytometry histograms for three maturation markers (CD80, PD-L1 and CD40) on pDCs and CD1c + mDCs. For CD1c: Light grey represents unstimulated samples, dark grey represents GM-CSF samples, and black represents pRNA samples. For pDC: bright transparent grey represents the unstimulated samples, light grey represents the IL-3 samples, grey represents FSME and black represents pRNA samples. B . Gene expression levels of the 3 donors of the RNA-seq. C . Correlation plot of the RNA-seq counts/1 million reads set out against the geometric mean fluorescence intensity as measured by flow cytometry (protein level). Throughout this paper, error bars represent standard error of the mean.

    Article Snippet: DCs were stimulated with: FSME (5%; Baxter AG, Vienna), pRNA (15μg/ml), CpG-P (5μg/ml; Miltenyi Biotech, Germany), GM-CSF (800 U/ml; (Cellgenix, Freiburg, Germany). pRNA complexes were prepared fresh 5-10 minutes before adding to the cell culture. pDCs were cultured with 10 ng/mL IL-3 (Cellgenix, Freiburg, Germany) as a survival factor in addition to the stimuli.

    Techniques: RNA Sequencing Assay, Flow Cytometry, Cytometry, Expressing, Fluorescence

    COMBIG/Ad5M-matured DCs express a mature phenotype and Th1-polarized cytokine secretion profile. (A) CD14 + monocytes were isolated from healthy donor PBMCs, differentiated into imDCs by GM-CSF/IL-4 for 5 days, matured under different conditions for 18 h, washed and further cultured for 24 h and analyzed. (B) DCs were characterized for HLA-DR, CD40, CD80, CD86 and CD83 expression by flow cytometry. Mean fluorescence intensity (MFI) for each marker on DCs (CD14 − CD1a + ) produced from eight donors are shown. (C) Secreted cytokines were assessed in supernatants of each treatment by proteome profiler where supernatants from six donors were pooled. (D) IL-12, IL-6 and CXCL10 secretion were also verified by ELISA for each donor. (E, F) Inflammasome activation was evaluated by the re-localization of the protein ASC, an inflammasome component, from a diffuse state to a single speck exhibited in representative FACS plots of ASC width (ASC-W) and ASC area (ASC-A) and % of speck + DCs produced from six donors. Data are shown as mean±SEM (n.s. p ≥ 0.05; * P

    Journal: Oncoimmunology

    Article Title: Pro-inflammatory allogeneic DCs promote activation of bystander immune cells and thereby license antigen-specific T-cell responses

    doi: 10.1080/2162402X.2017.1395126

    Figure Lengend Snippet: COMBIG/Ad5M-matured DCs express a mature phenotype and Th1-polarized cytokine secretion profile. (A) CD14 + monocytes were isolated from healthy donor PBMCs, differentiated into imDCs by GM-CSF/IL-4 for 5 days, matured under different conditions for 18 h, washed and further cultured for 24 h and analyzed. (B) DCs were characterized for HLA-DR, CD40, CD80, CD86 and CD83 expression by flow cytometry. Mean fluorescence intensity (MFI) for each marker on DCs (CD14 − CD1a + ) produced from eight donors are shown. (C) Secreted cytokines were assessed in supernatants of each treatment by proteome profiler where supernatants from six donors were pooled. (D) IL-12, IL-6 and CXCL10 secretion were also verified by ELISA for each donor. (E, F) Inflammasome activation was evaluated by the re-localization of the protein ASC, an inflammasome component, from a diffuse state to a single speck exhibited in representative FACS plots of ASC width (ASC-W) and ASC area (ASC-A) and % of speck + DCs produced from six donors. Data are shown as mean±SEM (n.s. p ≥ 0.05; * P

    Article Snippet: Monocytes isolated from PBMCs by CD14+ positive magnetic selection (Miltenyi Biotec) were differentiated to immature DCs (imDCs) using 20 ng/mL human IL-4 and 100 ng/mL GM-CSF (Gentaur) for 5 days.

    Techniques: Isolation, Cell Culture, Expressing, Flow Cytometry, Cytometry, Fluorescence, Marker, Produced, Enzyme-linked Immunosorbent Assay, Activation Assay, FACS

    Monocyte-derived dendritic cells and macrophages are barely sensitive for BV6-induced cell death. (A and B) Monocyte-derived macrophages, immature and Fc-CD40L maturated monocyte-derived dendritic cells were challenged for one day with and without 10 µM BV6. Cells were then analyzed by annexin-V staining for cell death induction (A; upper panel: representative analysis of one individual sample; lower panel: summary of the data of 3 independent DC experiments and 6 independent experiments with monocytes and macrophages). p100 processing were determined by western blotting and is shown for one representative experiment (B). (C) Monocytes cultivated overnight in GM-CSF/IL4, iDCs obtained after 7 days of cultivation with GM-CSF/IL4 and mDCs maturated with TNF or Fc-CD40L were analyzed by western blotting with respect to the expression of the indicated proteins (data shown are representative for four independent experiments).

    Journal: PLoS ONE

    Article Title: SMAC Mimetic BV6 Induces Cell Death in Monocytes and Maturation of Monocyte-Derived Dendritic Cells

    doi: 10.1371/journal.pone.0021556

    Figure Lengend Snippet: Monocyte-derived dendritic cells and macrophages are barely sensitive for BV6-induced cell death. (A and B) Monocyte-derived macrophages, immature and Fc-CD40L maturated monocyte-derived dendritic cells were challenged for one day with and without 10 µM BV6. Cells were then analyzed by annexin-V staining for cell death induction (A; upper panel: representative analysis of one individual sample; lower panel: summary of the data of 3 independent DC experiments and 6 independent experiments with monocytes and macrophages). p100 processing were determined by western blotting and is shown for one representative experiment (B). (C) Monocytes cultivated overnight in GM-CSF/IL4, iDCs obtained after 7 days of cultivation with GM-CSF/IL4 and mDCs maturated with TNF or Fc-CD40L were analyzed by western blotting with respect to the expression of the indicated proteins (data shown are representative for four independent experiments).

    Article Snippet: Monocytes were resuspended in RPMI-1460 supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin (PenStrep) (all PAA), 10 ng/ml IL4 (ImmunoTools GmbH, Friesoythe, Germany) and 50 ng/ml GM-CSF (ImmunoTools GmbH) and immediately prepared for the various experiments.

    Techniques: Derivative Assay, Staining, Western Blot, Expressing

    SMAC mimetic BV6 induces cell death in monocytes. (A) Monocytes were isolated from peripheral blood mononuclear cells by MACS separation, cultivated for 1 day in GM-CSF/IL4 supplemented medium in the presence and absence of 10 µM BV6 and finally visually inspected by microscopy. (B) Monocytes were cultivated with 10 µM BV6 for 1 day and viability was assessed using the MTT assay (triplicates, left panel). Viability of BV6 treated cells were normalized against corresponding control samples receiving no BV6 (n = 7, right panel). (C) Effect of 10 µM BV6 on monocyte viability was determined after overnight incubation by annexin-V staining. Left panel shows a representative analysis of one individual sample and the right panel summarizes the data of monocytes independently derived from 5 buffy coat samples. The mean is indicated by a horizontal line. (D) Freshly isolated monocytes (0 h) and monocytes cultivated overnight in GM-CSF/IL4 (24 h) were analyzed by western blotting with respect to the processing of the indicated caspases and PARP-1. (E and F) Freshly isolated monocytes and monocytes cultivated in the presence of the indicated mixtures of GM-CSF/IL4 and 10 µM BV6 were analyzed by western blotting (E) and FACS (F) with respect to the expression of the indicated proteins. The western blot data shown were representative for two – four independent experiments.

    Journal: PLoS ONE

    Article Title: SMAC Mimetic BV6 Induces Cell Death in Monocytes and Maturation of Monocyte-Derived Dendritic Cells

    doi: 10.1371/journal.pone.0021556

    Figure Lengend Snippet: SMAC mimetic BV6 induces cell death in monocytes. (A) Monocytes were isolated from peripheral blood mononuclear cells by MACS separation, cultivated for 1 day in GM-CSF/IL4 supplemented medium in the presence and absence of 10 µM BV6 and finally visually inspected by microscopy. (B) Monocytes were cultivated with 10 µM BV6 for 1 day and viability was assessed using the MTT assay (triplicates, left panel). Viability of BV6 treated cells were normalized against corresponding control samples receiving no BV6 (n = 7, right panel). (C) Effect of 10 µM BV6 on monocyte viability was determined after overnight incubation by annexin-V staining. Left panel shows a representative analysis of one individual sample and the right panel summarizes the data of monocytes independently derived from 5 buffy coat samples. The mean is indicated by a horizontal line. (D) Freshly isolated monocytes (0 h) and monocytes cultivated overnight in GM-CSF/IL4 (24 h) were analyzed by western blotting with respect to the processing of the indicated caspases and PARP-1. (E and F) Freshly isolated monocytes and monocytes cultivated in the presence of the indicated mixtures of GM-CSF/IL4 and 10 µM BV6 were analyzed by western blotting (E) and FACS (F) with respect to the expression of the indicated proteins. The western blot data shown were representative for two – four independent experiments.

    Article Snippet: Monocytes were resuspended in RPMI-1460 supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin (PenStrep) (all PAA), 10 ng/ml IL4 (ImmunoTools GmbH, Friesoythe, Germany) and 50 ng/ml GM-CSF (ImmunoTools GmbH) and immediately prepared for the various experiments.

    Techniques: Isolation, Magnetic Cell Separation, Microscopy, MTT Assay, Incubation, Staining, Derivative Assay, Western Blot, FACS, Expressing

    SMAC mimetic BV6 attenuates TNF- and CD40L-induced proinflammatory signaling and triggers DC maturation. (A and B) Immature monocyte-derived dendritic cells (iDCs) were generated by cultivation for 7 days with GM-CSF/IL4. To obtain mature dendritic cells (mDCs), iDCs were further treated with Fc-CD40L (200 ng/ml) for 2 days. mDCs were then cultivated in Fc-CD40L–free medium for additional 24 hours with and without 10 µM BV6 and were stimulated for the indicated times with TNF (200 ng/ml) and Fc-CD40L (200 ng/ml) (A). iDCs were primed overnight with 10 µM BV6 and were then challenged for 5 and 20 min with TNF (200 ng/ml) and Fc-CD40L (200 ng/ml) (B). iDCs and mDCs were finally analyzed by western blotting to determine the presence of the indicated proteins. The results shown with mDCs are representative of three independent experiments, the results with iDCs for two experiments. (C) iDCs were treated with 10 µM BV6, 300 ng/ml TNF and 1 µg/ml of Flag-CD40L oligomerized with 1 µg of the Flag-specific mAb M2 or as a control remained untreated. After three days cells were analyzed by FACS for the cell surface expression of CD83 and CD86. (upper panel: representative analysis of one individual sample; lower panel: summary of the data of iDCs of 4 independent donors. (D) Cell culture supernatants from “C” were analyzed for the presence of IL12. IL12 production was normalized to the corresponding values of untreated cells. The average of experiments with five independent donors is shown.

    Journal: PLoS ONE

    Article Title: SMAC Mimetic BV6 Induces Cell Death in Monocytes and Maturation of Monocyte-Derived Dendritic Cells

    doi: 10.1371/journal.pone.0021556

    Figure Lengend Snippet: SMAC mimetic BV6 attenuates TNF- and CD40L-induced proinflammatory signaling and triggers DC maturation. (A and B) Immature monocyte-derived dendritic cells (iDCs) were generated by cultivation for 7 days with GM-CSF/IL4. To obtain mature dendritic cells (mDCs), iDCs were further treated with Fc-CD40L (200 ng/ml) for 2 days. mDCs were then cultivated in Fc-CD40L–free medium for additional 24 hours with and without 10 µM BV6 and were stimulated for the indicated times with TNF (200 ng/ml) and Fc-CD40L (200 ng/ml) (A). iDCs were primed overnight with 10 µM BV6 and were then challenged for 5 and 20 min with TNF (200 ng/ml) and Fc-CD40L (200 ng/ml) (B). iDCs and mDCs were finally analyzed by western blotting to determine the presence of the indicated proteins. The results shown with mDCs are representative of three independent experiments, the results with iDCs for two experiments. (C) iDCs were treated with 10 µM BV6, 300 ng/ml TNF and 1 µg/ml of Flag-CD40L oligomerized with 1 µg of the Flag-specific mAb M2 or as a control remained untreated. After three days cells were analyzed by FACS for the cell surface expression of CD83 and CD86. (upper panel: representative analysis of one individual sample; lower panel: summary of the data of iDCs of 4 independent donors. (D) Cell culture supernatants from “C” were analyzed for the presence of IL12. IL12 production was normalized to the corresponding values of untreated cells. The average of experiments with five independent donors is shown.

    Article Snippet: Monocytes were resuspended in RPMI-1460 supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin (PenStrep) (all PAA), 10 ng/ml IL4 (ImmunoTools GmbH, Friesoythe, Germany) and 50 ng/ml GM-CSF (ImmunoTools GmbH) and immediately prepared for the various experiments.

    Techniques: Derivative Assay, Generated, Western Blot, FACS, Expressing, Cell Culture

    GM-CSF and IL4 rapidly induce resistance against BV6-induced cell death in monocytes. (A and B) Monocytes were cultivated in medium supplemented with GM-CSF/IL4 and were treated immediately or after 1 and 2 days with 10 µM BV6. One day after stimulation BV6 treated cells and a corresponding control sample cultivated without BV6 were analyzed by annexin-V staining (A) and western blotting (B).

    Journal: PLoS ONE

    Article Title: SMAC Mimetic BV6 Induces Cell Death in Monocytes and Maturation of Monocyte-Derived Dendritic Cells

    doi: 10.1371/journal.pone.0021556

    Figure Lengend Snippet: GM-CSF and IL4 rapidly induce resistance against BV6-induced cell death in monocytes. (A and B) Monocytes were cultivated in medium supplemented with GM-CSF/IL4 and were treated immediately or after 1 and 2 days with 10 µM BV6. One day after stimulation BV6 treated cells and a corresponding control sample cultivated without BV6 were analyzed by annexin-V staining (A) and western blotting (B).

    Article Snippet: Monocytes were resuspended in RPMI-1460 supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin (PenStrep) (all PAA), 10 ng/ml IL4 (ImmunoTools GmbH, Friesoythe, Germany) and 50 ng/ml GM-CSF (ImmunoTools GmbH) and immediately prepared for the various experiments.

    Techniques: Staining, Western Blot

    BV6 induces apoptotic and necrotic cell death in monocytes. (A) Freshly isolated monocytes were incubated in GM-CSF/IL4 supplemented medium for 1 h with the indicated mixtures of z-VAD-fmk (20 µM), necrostatin-1 (70 µM) and the TNF inhibitor TNFR2-Fc/Enbrel (50 µg/ml). Cells were then challenged overnight with 10 µM BV6 and cell viability was finally evaluated by help of the MTT assay. (B) Freshly isolated monocytes and monocytes cultivated overnight in the presence of the indicated mixtures of GM-CSF/IL4 and 10 µM BV6 were analyzed by FACS for cell surface expression of membrane TNF and the death receptors TNFR1, CD95 and TRAILR2. (C) Monocytes were challenged with BV6 in the presence of soluble Fc fusion proteins of TRAILR2 (5 µg/ml), CD95 (50 µg/ml) and TNFR2 (Enbrel, 20 µg/ml) or a mixture of them. After 24 h viability was determined using the MTT assay (Left panel). The functionality of the three Fc fusion proteins was controlled in cell death assays with HT1080 and recombinant 50 ng/ml TNF, 4 ng/ml Fc-CD95L and 50 ng/mlTRAIL (right panel). Data shown are representative for three independent experiments.

    Journal: PLoS ONE

    Article Title: SMAC Mimetic BV6 Induces Cell Death in Monocytes and Maturation of Monocyte-Derived Dendritic Cells

    doi: 10.1371/journal.pone.0021556

    Figure Lengend Snippet: BV6 induces apoptotic and necrotic cell death in monocytes. (A) Freshly isolated monocytes were incubated in GM-CSF/IL4 supplemented medium for 1 h with the indicated mixtures of z-VAD-fmk (20 µM), necrostatin-1 (70 µM) and the TNF inhibitor TNFR2-Fc/Enbrel (50 µg/ml). Cells were then challenged overnight with 10 µM BV6 and cell viability was finally evaluated by help of the MTT assay. (B) Freshly isolated monocytes and monocytes cultivated overnight in the presence of the indicated mixtures of GM-CSF/IL4 and 10 µM BV6 were analyzed by FACS for cell surface expression of membrane TNF and the death receptors TNFR1, CD95 and TRAILR2. (C) Monocytes were challenged with BV6 in the presence of soluble Fc fusion proteins of TRAILR2 (5 µg/ml), CD95 (50 µg/ml) and TNFR2 (Enbrel, 20 µg/ml) or a mixture of them. After 24 h viability was determined using the MTT assay (Left panel). The functionality of the three Fc fusion proteins was controlled in cell death assays with HT1080 and recombinant 50 ng/ml TNF, 4 ng/ml Fc-CD95L and 50 ng/mlTRAIL (right panel). Data shown are representative for three independent experiments.

    Article Snippet: Monocytes were resuspended in RPMI-1460 supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin (PenStrep) (all PAA), 10 ng/ml IL4 (ImmunoTools GmbH, Friesoythe, Germany) and 50 ng/ml GM-CSF (ImmunoTools GmbH) and immediately prepared for the various experiments.

    Techniques: Isolation, Incubation, MTT Assay, FACS, Expressing, Recombinant

    Effect of internalized p41 fragment on the proteolytic activity of cysteine proteases (A) and the secretion of IL-12/p70 (B, C). Samples (A): cell lysates of non-treated immature DC, immature DC after a 6-h incubation with p41 fragment (0.035 μM, 0.35 μM and 3.5 μM) and non-treated immature DC with 10 μM E-64. Fluorogenic substrate in buffer with DTT was used as blank (BLK). Representative measurements, each of three biological repetitions, are shown. Samples (B and C): cell free supernatants (culture media) of immature DC, preincubated with p41 fragment (0.035 μM, 0.35 μM and 3.5 μM) for 6 h prior to their maturation with TNF-α (B) or LPS (C). Non-treated cells (no preincubation with p41 fragment): immature DC, cultured in the presence of GM-CSF for three days (no maturation), DC matured with TNF-α, and DC matured with LPS. Pretreated but non-matured cells: immature DC, pretreated with 3.5 μM p41 fragment, and cultured in the presence of GM-CSF. IL-12 concentrations (in pg/ml) were measured in triplicate, average values ± SD are shown. (D) Immunolabelled cathepsins L and S in DC lysates. Samples (50 μg per well): (1) immature DC, (2) DC pretreated with 3.5 μM p41 fragment for 6 h, no LPS, (3) DC matured with LPS for 24 h, (4) DC pretreated with 3.5 μM p41 fragment for 6 h and matured with LPS for 24 h.

    Journal: PLoS ONE

    Article Title: Exogenous Thyropin from p41 Invariant Chain Diminishes Cysteine Protease Activity and Affects IL-12 Secretion during Maturation of Human Dendritic Cells

    doi: 10.1371/journal.pone.0150815

    Figure Lengend Snippet: Effect of internalized p41 fragment on the proteolytic activity of cysteine proteases (A) and the secretion of IL-12/p70 (B, C). Samples (A): cell lysates of non-treated immature DC, immature DC after a 6-h incubation with p41 fragment (0.035 μM, 0.35 μM and 3.5 μM) and non-treated immature DC with 10 μM E-64. Fluorogenic substrate in buffer with DTT was used as blank (BLK). Representative measurements, each of three biological repetitions, are shown. Samples (B and C): cell free supernatants (culture media) of immature DC, preincubated with p41 fragment (0.035 μM, 0.35 μM and 3.5 μM) for 6 h prior to their maturation with TNF-α (B) or LPS (C). Non-treated cells (no preincubation with p41 fragment): immature DC, cultured in the presence of GM-CSF for three days (no maturation), DC matured with TNF-α, and DC matured with LPS. Pretreated but non-matured cells: immature DC, pretreated with 3.5 μM p41 fragment, and cultured in the presence of GM-CSF. IL-12 concentrations (in pg/ml) were measured in triplicate, average values ± SD are shown. (D) Immunolabelled cathepsins L and S in DC lysates. Samples (50 μg per well): (1) immature DC, (2) DC pretreated with 3.5 μM p41 fragment for 6 h, no LPS, (3) DC matured with LPS for 24 h, (4) DC pretreated with 3.5 μM p41 fragment for 6 h and matured with LPS for 24 h.

    Article Snippet: Immature DC (1×106 cells/ml) were matured, either with TNF-α (15 ng/ml, R & D Systems) and GM-CSF (Leucomax, 1000 U/ml, Novartis Pharma) for three to five days or with LPS (20 ng/ml, Sigma-Aldrich) and GM-CSF (Leucomax, 500 U/ml, Novartis Pharma) for up to 48 h. Cell viability was checked using trypan blue (Sigma-Aldrich).

    Techniques: Activity Assay, Incubation, Cell Culture

    IFN-γ and GM-CSF production of IL-15- and/or IL-18-stimulated NK cells and effects of commercial and S. griseus 10/ppi valinomycins on cytokine production. The stimulatory effects of IL-15 and IL-18 on cytokine production are synergistic. Valinomycin almost completely inhibits IL-15-induced cytokine production, whereas there is some cytokine production left in cells activated by a combination of IL-15 and IL-18 and pretreated with valinomycin. The P values in paired t tests for the differences between IFN-γ production by lymphocytes treated with IL-15 with valinomycin and that by lymphocytes treated with IL-15 and IL-18 with valinomycin are

    Journal: Infection and Immunity

    Article Title: Inhibition of Human NK Cell Function by Valinomycin, a Toxin from Streptomyces griseus in Indoor Air

    doi:

    Figure Lengend Snippet: IFN-γ and GM-CSF production of IL-15- and/or IL-18-stimulated NK cells and effects of commercial and S. griseus 10/ppi valinomycins on cytokine production. The stimulatory effects of IL-15 and IL-18 on cytokine production are synergistic. Valinomycin almost completely inhibits IL-15-induced cytokine production, whereas there is some cytokine production left in cells activated by a combination of IL-15 and IL-18 and pretreated with valinomycin. The P values in paired t tests for the differences between IFN-γ production by lymphocytes treated with IL-15 with valinomycin and that by lymphocytes treated with IL-15 and IL-18 with valinomycin are

    Article Snippet: The culture supernatants were harvested, and the cytokine concentrations were determined by enzyme-linked immunosorbent assay using paired antibodies for IFN-γ (Diaclone, Besançon, France) and GM-CSF (PharMingen, San Diego, Calif.).

    Techniques:

    The effect of cytokines on the development of CR3 + and CR4 + macrophages and in MDM. ( a ) Monocytes were treated with 40 ng/ml LT-α, IFN-γ, IL-4, IL-13, IL-1β, IL-6, IL-10, M-CSF, GM-CSF or dexamethasone, 20 ng/ml TNF, 15 ng/ml TGF-β1. ( b ) Monocytes were cultured for 3 days for maturation into macrophages. The MDM were then incubated for 24 h with 40 ng/ml of the cytokines, LT-α, IFN-γ, IL-4, IL-1β, IL-6, IL-10, IL-13, M-CSF, GM-CSF, 20 ng/ml TNF, 15 ng/ml TGF-β1 or Dexamethasone (50 ng/ml). The level of CD11b and CD11c mRNA was measured using qPCR. Data are expressed as fold-change over GAPDH-normalized CD11b and CD11c mRNA in the absence of cytokine set as 1. Data are presented as means ± SD of three experiments, each conducted with cells from different individuals, *p

    Journal: Scientific Reports

    Article Title: Cytokines regulate complement receptor immunoglobulin expression and phagocytosis of Candida albicans in human macrophages: A control point in anti-microbial immunity

    doi: 10.1038/s41598-017-04325-0

    Figure Lengend Snippet: The effect of cytokines on the development of CR3 + and CR4 + macrophages and in MDM. ( a ) Monocytes were treated with 40 ng/ml LT-α, IFN-γ, IL-4, IL-13, IL-1β, IL-6, IL-10, M-CSF, GM-CSF or dexamethasone, 20 ng/ml TNF, 15 ng/ml TGF-β1. ( b ) Monocytes were cultured for 3 days for maturation into macrophages. The MDM were then incubated for 24 h with 40 ng/ml of the cytokines, LT-α, IFN-γ, IL-4, IL-1β, IL-6, IL-10, IL-13, M-CSF, GM-CSF, 20 ng/ml TNF, 15 ng/ml TGF-β1 or Dexamethasone (50 ng/ml). The level of CD11b and CD11c mRNA was measured using qPCR. Data are expressed as fold-change over GAPDH-normalized CD11b and CD11c mRNA in the absence of cytokine set as 1. Data are presented as means ± SD of three experiments, each conducted with cells from different individuals, *p

    Article Snippet: Cytokines and cell culture reagents Recombinant granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage (M)-CSF, interleukin (IL)-1β, IL-6, IL-4, IL-10, IL-13, interferon (IFN)-γ, lymphotoxin (LT)-α, tumor necrosis factor (TNF), M-CSF and GM-CSF were purchased from ProSpec-Tany Technogene (Rehovot, Israel), transforming growth factor (TGF)-β1 from R & D Systems (Minneapolis, MN), and dexamethasone was purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques: Cell Culture, Incubation, Real-time Polymerase Chain Reaction

    Effects of cytokines on the phagocytosis of C . albicans by MDM. MDM were prepared by culturing human monocytes for 5 days. The MDM were treated with 40 ng/ml of LT-α ( a ), IFN-γ ( b ), IL-4 ( c ), IL-13 ( d ), or 20 ng/ml TNF ( e ), or 40 ng/ml IL-1β ( f ), IL-6 ( g ), or 15 ng/ml TGF-β1 ( h ) or 40 ng/ml IL-10 ( i ), M-CSF/GM-CSF ( j , k ) or 50 ng/ml dexamethasone ( l ) for 24 h and examined for their ability to phagocytose complement opsonised C . albicans . Phagocytosis was scored as both the number of macrophages that had engulfed more than > 4 fungi (line graph) and the number of fungi engulfed per cell (bar graph). Data are presented as means ± SD of three experiments, each conducted with cells from different individuals, *p

    Journal: Scientific Reports

    Article Title: Cytokines regulate complement receptor immunoglobulin expression and phagocytosis of Candida albicans in human macrophages: A control point in anti-microbial immunity

    doi: 10.1038/s41598-017-04325-0

    Figure Lengend Snippet: Effects of cytokines on the phagocytosis of C . albicans by MDM. MDM were prepared by culturing human monocytes for 5 days. The MDM were treated with 40 ng/ml of LT-α ( a ), IFN-γ ( b ), IL-4 ( c ), IL-13 ( d ), or 20 ng/ml TNF ( e ), or 40 ng/ml IL-1β ( f ), IL-6 ( g ), or 15 ng/ml TGF-β1 ( h ) or 40 ng/ml IL-10 ( i ), M-CSF/GM-CSF ( j , k ) or 50 ng/ml dexamethasone ( l ) for 24 h and examined for their ability to phagocytose complement opsonised C . albicans . Phagocytosis was scored as both the number of macrophages that had engulfed more than > 4 fungi (line graph) and the number of fungi engulfed per cell (bar graph). Data are presented as means ± SD of three experiments, each conducted with cells from different individuals, *p

    Article Snippet: Cytokines and cell culture reagents Recombinant granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage (M)-CSF, interleukin (IL)-1β, IL-6, IL-4, IL-10, IL-13, interferon (IFN)-γ, lymphotoxin (LT)-α, tumor necrosis factor (TNF), M-CSF and GM-CSF were purchased from ProSpec-Tany Technogene (Rehovot, Israel), transforming growth factor (TGF)-β1 from R & D Systems (Minneapolis, MN), and dexamethasone was purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques:

    Effects of cytokines on CRIg expression in matured macrophages (MDM). In these studies MDM were prepared by culturing human monocytes for 3 days. MDM from 3 day cultures were treated with LT-α ( a ) (0, 5, 10, 20 and 40 ng/ml) or IFN-γ ( b ) (0, 5, 20 and 40 ng/ml) or IL-4 ( c ) (0, 1, 3, 5, 10 and 40 ng/ml) or IL-13 ( d ) (0, 5, 10, 20 and 40 ng/ml), TGF-β1 ( e ) (0, 2, 5 and 15 ng/ml) or ( f ) IL-10 (0, 5, 10, 20 and 40 ng/ml) or M-CSF/GM-CSF ( g , h ) (0, 5, 10, 20 and 40 ng/ml) or dexamethasone ( i ) (0, 10, 30 and 50 ng/ml) for 24 h and then CRIg mRNA levels relative to GAPDH mRNA were assessed by qPCR. Data are expressed as fold-change over GAPDH-normalised CRIg mRNA in the absence of cytokine set as 1. Data are presented as means ± SD of three experiments, each conducted with cells from different individuals, *p

    Journal: Scientific Reports

    Article Title: Cytokines regulate complement receptor immunoglobulin expression and phagocytosis of Candida albicans in human macrophages: A control point in anti-microbial immunity

    doi: 10.1038/s41598-017-04325-0

    Figure Lengend Snippet: Effects of cytokines on CRIg expression in matured macrophages (MDM). In these studies MDM were prepared by culturing human monocytes for 3 days. MDM from 3 day cultures were treated with LT-α ( a ) (0, 5, 10, 20 and 40 ng/ml) or IFN-γ ( b ) (0, 5, 20 and 40 ng/ml) or IL-4 ( c ) (0, 1, 3, 5, 10 and 40 ng/ml) or IL-13 ( d ) (0, 5, 10, 20 and 40 ng/ml), TGF-β1 ( e ) (0, 2, 5 and 15 ng/ml) or ( f ) IL-10 (0, 5, 10, 20 and 40 ng/ml) or M-CSF/GM-CSF ( g , h ) (0, 5, 10, 20 and 40 ng/ml) or dexamethasone ( i ) (0, 10, 30 and 50 ng/ml) for 24 h and then CRIg mRNA levels relative to GAPDH mRNA were assessed by qPCR. Data are expressed as fold-change over GAPDH-normalised CRIg mRNA in the absence of cytokine set as 1. Data are presented as means ± SD of three experiments, each conducted with cells from different individuals, *p

    Article Snippet: Cytokines and cell culture reagents Recombinant granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage (M)-CSF, interleukin (IL)-1β, IL-6, IL-4, IL-10, IL-13, interferon (IFN)-γ, lymphotoxin (LT)-α, tumor necrosis factor (TNF), M-CSF and GM-CSF were purchased from ProSpec-Tany Technogene (Rehovot, Israel), transforming growth factor (TGF)-β1 from R & D Systems (Minneapolis, MN), and dexamethasone was purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    The EphA2 receptor tyrosine kinase is expressed by in vitro generated LLDC Adherent mononuclear cells were isolated and cultured one week in the presence of GM-CSF and IL-4 (for monocyte derived dendritic cells, DC) and TGF-β (for monocyte derived LLDC). (A) Cells were incubated with a monoclonal EphA2 antibody (bold line) or an irrelevant antibody (dotted line) followed by staining with PE-labeled α-mouse antibody. (B) Cells were incubated with a soluble EphA2 ligand fusion protein (ephrin-A4-Fc, bold line) and a negative control fusion protein (CD19-Fc, dotted line). (C) A Western blot with cell lysates from monocyte derived dendritic cells (DC) or LLDC (LLDC) were immunoblotted using a polyclonal anti-EphA2 antiserum.

    Journal: BMC Immunology

    Article Title: Expression and functional effects of Eph receptor tyrosine kinase A family members on Langerhans like dendritic cells

    doi: 10.1186/1471-2172-5-9

    Figure Lengend Snippet: The EphA2 receptor tyrosine kinase is expressed by in vitro generated LLDC Adherent mononuclear cells were isolated and cultured one week in the presence of GM-CSF and IL-4 (for monocyte derived dendritic cells, DC) and TGF-β (for monocyte derived LLDC). (A) Cells were incubated with a monoclonal EphA2 antibody (bold line) or an irrelevant antibody (dotted line) followed by staining with PE-labeled α-mouse antibody. (B) Cells were incubated with a soluble EphA2 ligand fusion protein (ephrin-A4-Fc, bold line) and a negative control fusion protein (CD19-Fc, dotted line). (C) A Western blot with cell lysates from monocyte derived dendritic cells (DC) or LLDC (LLDC) were immunoblotted using a polyclonal anti-EphA2 antiserum.

    Article Snippet: IL-4 was a kind gift from Schering-Plough Research Institute (Kenilworth, J), TGF-β1 was obtained from Habersham Pharmacia Biotech (Uppsala, Sweden) and GM-CSF was obtained from Roche (Mannheim, Germany).

    Techniques: In Vitro, Generated, Isolation, Cell Culture, Derivative Assay, Incubation, Staining, Labeling, Negative Control, Western Blot

    Profiling of antibody responses in cancer patients to CTLA-4 blockade and GM-CSF with protein microarrays

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

    Article Title: Diversity of antigen-specific responses induced in vivo with CTLA-4 blockade in prostate cancer patients

    doi: 10.4049/jimmunol.1201529

    Figure Lengend Snippet: Profiling of antibody responses in cancer patients to CTLA-4 blockade and GM-CSF with protein microarrays

    Article Snippet: A phase I/II trial combined escalating doses of anti-CTLA-4 antibody (ipilimumab, Bristol-Myer Squibb) with a fixed dose of GM-CSF (sargramostim, Sanofi) was performed to assess for safety, feasibility, and immunogenicity in patients with CRPC ( ) ( ).

    Techniques:

    Tumor environmental factors turn monocytes into CD163 high CD86 low IL‐10 high MΦ. HD CD14 + monocytes were cultured in the presence of 25% SNDils for 7 days, and surface markers and cytokine production were evaluated 24 h after addition of LPS. (a) r‐CD163 MFI from control APCs ( n = 8 independent donors) and SNDil‐MΦ ( n = 29 independent SNDils). (b) Representative pseudocolour plots of CD64 + CD163 + cells of M0‐MΦ and SNDil‐MΦ based on the median obtained within each group. (c) Expression of CD86 and (d) production of TNF‐α and IL‐10 in CD163 low and CD163 high SNDil‐MΦ or in control APCs. For (a–d) , experiments performed with, at least, five independent donor monocytes and different SNDils ( n = 29). (e) Correlation of r‐CD163 MFI and IL‐10 production among SNDil‐MΦ. (f, g) SNDil‐MΦ were cultured in the presence of neutralising anti‐M‐CSF, anti‐TGF‐β and anti‐VEGF or control antibodies during the differentiation process and were activated by LPS during the last 24 h to evaluate surface markers (f) and cytokine production (g) in CD163 high IL‐10 high SNDil‐MΦ (red dots, n = 4; * P ≤ 0.05) and CD163 low IL‐10 low SNDil‐MΦ (blue dots; n = 3).

    Journal: Clinical & Translational Immunology

    Article Title: CD163+ tumor‐associated macrophage accumulation in breast cancer patients reflects both local differentiation signals and systemic skewing of monocytes

    doi: 10.1002/cti2.1108

    Figure Lengend Snippet: Tumor environmental factors turn monocytes into CD163 high CD86 low IL‐10 high MΦ. HD CD14 + monocytes were cultured in the presence of 25% SNDils for 7 days, and surface markers and cytokine production were evaluated 24 h after addition of LPS. (a) r‐CD163 MFI from control APCs ( n = 8 independent donors) and SNDil‐MΦ ( n = 29 independent SNDils). (b) Representative pseudocolour plots of CD64 + CD163 + cells of M0‐MΦ and SNDil‐MΦ based on the median obtained within each group. (c) Expression of CD86 and (d) production of TNF‐α and IL‐10 in CD163 low and CD163 high SNDil‐MΦ or in control APCs. For (a–d) , experiments performed with, at least, five independent donor monocytes and different SNDils ( n = 29). (e) Correlation of r‐CD163 MFI and IL‐10 production among SNDil‐MΦ. (f, g) SNDil‐MΦ were cultured in the presence of neutralising anti‐M‐CSF, anti‐TGF‐β and anti‐VEGF or control antibodies during the differentiation process and were activated by LPS during the last 24 h to evaluate surface markers (f) and cytokine production (g) in CD163 high IL‐10 high SNDil‐MΦ (red dots, n = 4; * P ≤ 0.05) and CD163 low IL‐10 low SNDil‐MΦ (blue dots; n = 3).

    Article Snippet: Isolated monocytes were then differentiated in complete RPMI‐1640 medium (containing antibiotics and 10% heat‐inactivated FCS) for 7 days into the different control APC populations, that is: cultured in medium alone for M0‐MΦ; GM‐CSF (50 ng mL−1 , Schering‐Plough, Dardilly, France) and IFN‐γ (20 ng mL−1 , PeproTech, Neuilly‐sur‐Seine, France) for M1‐MΦ; M‐CSF (50 ng mL−1 , R & D Systems, Lille, France) and IL‐4 (20 ng mL−1 , Schering‐Plough) for M2‐MΦ; and GM‐CSF (50 ng mL−1 ) and IL‐4 (20 ng mL−1 ) for Mo‐DC.

    Techniques: Cell Culture, Expressing

    Monocyte genes induced by both GM-CSF and 15 kDa granulysin . Hierarchical clustering of the 3191, 2416, 1534 and 1738 genes induced by both factors at 4 (a), 12 (b), 24 (c) and 48 (d) hours respectively (p-value

    Journal: Journal of Translational Medicine

    Article Title: 15 kDa Granulysin versus GM-CSF for monocytes differentiation: analogies and differences at the transcriptome level

    doi: 10.1186/1479-5876-9-41

    Figure Lengend Snippet: Monocyte genes induced by both GM-CSF and 15 kDa granulysin . Hierarchical clustering of the 3191, 2416, 1534 and 1738 genes induced by both factors at 4 (a), 12 (b), 24 (c) and 48 (d) hours respectively (p-value

    Article Snippet: Fresh monocytes were cultured in 6-well plates (Corning Costar, Corning Incorporated, Corning, NY, USA) at a concentration of 2 ×10 6 cell/ml in 90% RPMI-1640 media, 10% AB heat inactivated plasma, 10 mcg/ml gentamicin in the presence of 15 kDa Granulysin (10 nM) or GM-CSF (Leukine Sagramostin, 10 ng/ml, 56 IU/ml, Genzyme, Cambridge, MA, USA) and harvested at 4, 12, 24 and 48 hours.

    Techniques: Significance Assay

    Gene expression analysis of monocytes cultured with GM-CSF or 15 kDa Granulysin . a) Principal component analysis of all samples based on the entire dataset (33757 genes); b) Dendrogram of the unsupervised cluster of 9951 genes that were present in at least 22 samples and whose expression differed in at least 5 samples by more than 1.75-fold from the median

    Journal: Journal of Translational Medicine

    Article Title: 15 kDa Granulysin versus GM-CSF for monocytes differentiation: analogies and differences at the transcriptome level

    doi: 10.1186/1479-5876-9-41

    Figure Lengend Snippet: Gene expression analysis of monocytes cultured with GM-CSF or 15 kDa Granulysin . a) Principal component analysis of all samples based on the entire dataset (33757 genes); b) Dendrogram of the unsupervised cluster of 9951 genes that were present in at least 22 samples and whose expression differed in at least 5 samples by more than 1.75-fold from the median

    Article Snippet: Fresh monocytes were cultured in 6-well plates (Corning Costar, Corning Incorporated, Corning, NY, USA) at a concentration of 2 ×10 6 cell/ml in 90% RPMI-1640 media, 10% AB heat inactivated plasma, 10 mcg/ml gentamicin in the presence of 15 kDa Granulysin (10 nM) or GM-CSF (Leukine Sagramostin, 10 ng/ml, 56 IU/ml, Genzyme, Cambridge, MA, USA) and harvested at 4, 12, 24 and 48 hours.

    Techniques: Expressing, Cell Culture