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  • 99
    Vector Laboratories fluorescein avidin dcs
    Differences in sialylation between parental, non-invasive LNCaP and bone metastatic C4-2B cells ( A ) Flow cytometric determination of increased cell surface expression levels of α2,3- (left panel) and equal expression levels of α2,6- (right panel) linked sialic acid residues in C4-2B cells, using the lectins MAA and <t>SNA,</t> respectively. Single cell suspensions of LNCaP and C4-2B cells were incubated with biotinylated-MAA and SNA, followed by Fluorescein Avidin <t>DCS</t> and analysed on the Cell Lab Quanta SC MPL, stainings without the particular lectins were used as controls. Each experiment was performed at least three times. ( B ) Lectin blot analysis for total expression levels of α2,3- and α2,6-linked sialic acids in LNCaP and C4-2B cell lysates, containing 25 μg protein. Lysates were analysed by 4–20% gradient or 7.5% SDS/PAGE, and blotted with MAA (left panel) and SNA (right panel). Sialidase treatment (0.5 U/ml sodium citrate buffer at pH 6) of the blotted membranes for 16 h at 37°C, serves as control for the presence of sialic acid residues. Scion Image densitometry analysis of bands indicating the presence of α2,3- and α2,6-linked sialic acids in both cell lines (lower panels) and evaluation of the increased density at 125 kDa in C4-2B cells (side left panel) ( C ) Transcriptional expression levels of ST genes resulting in α2,3- and α2,6-linked sialic acid residues. Expression levels were determined by QPCR, normalized against HPRT (hypoxanthine–guanine phosphoribosyltransferase) and levels present in C4-2B cells are expressed relative as compared with the expression in the parental, non-invasive LNCaP cells. Analysed and evaluated data are means±S.D. from at least three independent experiments, asterisks indicate statistical difference from parental, non-invasive LNCaP control cells ( P
    Fluorescein Avidin Dcs, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 503 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Bio-Rad dc protein assay kit
    Effect of morphine on HCV NS5 <t>protein</t> expression in Huh.8 ( A ) and FCA-1 ( B ) cells. Huh.8 and FCA-1 cells (10 5 cells/well) plated in a 24-well plate were incubated with or without (Control) morphine at 10 −6 mol/L for 72 hours. Cell lysates were quantified by <t>DC</t> protein <t>assay</t> <t>kit.</t> Equal amounts (0.5 μg) of protein extracted from treated and untreated Huh.8 and FCA-1 cells were applied onto a NC membrane for immunoblot assay. The results were recorded on the film (2 minutes exposure). One representative result from three experiments is shown.
    Dc Protein Assay Kit, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 96/100, based on 29391 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Milliken des jarlais dc
    Effect of morphine on HCV NS5 <t>protein</t> expression in Huh.8 ( A ) and FCA-1 ( B ) cells. Huh.8 and FCA-1 cells (10 5 cells/well) plated in a 24-well plate were incubated with or without (Control) morphine at 10 −6 mol/L for 72 hours. Cell lysates were quantified by <t>DC</t> protein <t>assay</t> <t>kit.</t> Equal amounts (0.5 μg) of protein extracted from treated and untreated Huh.8 and FCA-1 cells were applied onto a NC membrane for immunoblot assay. The results were recorded on the film (2 minutes exposure). One representative result from three experiments is shown.
    Des Jarlais Dc, supplied by Milliken, used in various techniques. Bioz Stars score: 91/100, based on 265 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Carville Ltd montefiori dc
    Effect of morphine on HCV NS5 <t>protein</t> expression in Huh.8 ( A ) and FCA-1 ( B ) cells. Huh.8 and FCA-1 cells (10 5 cells/well) plated in a 24-well plate were incubated with or without (Control) morphine at 10 −6 mol/L for 72 hours. Cell lysates were quantified by <t>DC</t> protein <t>assay</t> <t>kit.</t> Equal amounts (0.5 μg) of protein extracted from treated and untreated Huh.8 and FCA-1 cells were applied onto a NC membrane for immunoblot assay. The results were recorded on the film (2 minutes exposure). One representative result from three experiments is shown.
    Montefiori Dc, supplied by Carville Ltd, used in various techniques. Bioz Stars score: 91/100, based on 263 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Assay Biotechnology dc protein assay
    Effect of morphine on HCV NS5 <t>protein</t> expression in Huh.8 ( A ) and FCA-1 ( B ) cells. Huh.8 and FCA-1 cells (10 5 cells/well) plated in a 24-well plate were incubated with or without (Control) morphine at 10 −6 mol/L for 72 hours. Cell lysates were quantified by <t>DC</t> protein <t>assay</t> <t>kit.</t> Equal amounts (0.5 μg) of protein extracted from treated and untreated Huh.8 and FCA-1 cells were applied onto a NC membrane for immunoblot assay. The results were recorded on the film (2 minutes exposure). One representative result from three experiments is shown.
    Dc Protein Assay, supplied by Assay Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 936 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    InvivoGen 5 aza dc
    <t>5-Aza-2′-deoxycytidine</t> (5-aza-dC)-mediated induction of Foxp3 expression in CD8 + T cells in vitro . Naive CD4 + and CD8 + T cells (both Foxp3 GFP− CD25 − CD62L high ) were FACS-isolated to high purity from pooled lymph nodes and spleen of adult Foxp3 GFP mice and subjected to T cell receptor stimulation in vitro (anti-CD3/CD28-coated beads) in the presence of IL-2 (100 U/ml), either alone or with added TGF-β (0.5 ng/ml) and/or 5-aza-dC (0.5 µM), as indicated. Cultures were analyzed at day 3 for Foxp3 GFP and CD25 expression among gated CD4 + or CD8 + T cells. (A) Representative flow cytometry of Foxp3 GFP /CD25 expression among gated CD4 + (top, black) and CD8 + (bottom, blue) T cells. Numbers in dot plots indicate the percentages of cells within the respective quadrant. (B,C) Median fluorescence intensities (MFIs) of CD25 expression among gated (B) CD4 + T cells (left: Foxp3 GFP− , right: Foxp3 GFP+ ) and (C) CD8 + T cells (left: Foxp3 GFP− , right: Foxp3 GFP+ ). Graphs show mean percentages ±SD of CD25 MFI from triplicate wells. The level of significance was determined by two-way ANOVA with Tukey’s multiple comparison test: **** P ≤ 0.0001 and P > 0.05 (ns).
    5 Aza Dc, supplied by InvivoGen, used in various techniques. Bioz Stars score: 92/100, based on 151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    brain products gmbh brainamp dc amplifier
    <t>5-Aza-2′-deoxycytidine</t> (5-aza-dC)-mediated induction of Foxp3 expression in CD8 + T cells in vitro . Naive CD4 + and CD8 + T cells (both Foxp3 GFP− CD25 − CD62L high ) were FACS-isolated to high purity from pooled lymph nodes and spleen of adult Foxp3 GFP mice and subjected to T cell receptor stimulation in vitro (anti-CD3/CD28-coated beads) in the presence of IL-2 (100 U/ml), either alone or with added TGF-β (0.5 ng/ml) and/or 5-aza-dC (0.5 µM), as indicated. Cultures were analyzed at day 3 for Foxp3 GFP and CD25 expression among gated CD4 + or CD8 + T cells. (A) Representative flow cytometry of Foxp3 GFP /CD25 expression among gated CD4 + (top, black) and CD8 + (bottom, blue) T cells. Numbers in dot plots indicate the percentages of cells within the respective quadrant. (B,C) Median fluorescence intensities (MFIs) of CD25 expression among gated (B) CD4 + T cells (left: Foxp3 GFP− , right: Foxp3 GFP+ ) and (C) CD8 + T cells (left: Foxp3 GFP− , right: Foxp3 GFP+ ). Graphs show mean percentages ±SD of CD25 MFI from triplicate wells. The level of significance was determined by two-way ANOVA with Tukey’s multiple comparison test: **** P ≤ 0.0001 and P > 0.05 (ns).
    Brainamp Dc Amplifier, supplied by brain products gmbh, used in various techniques. Bioz Stars score: 99/100, based on 159 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    SCHNEEBERGER meyer dc
    <t>5-Aza-2′-deoxycytidine</t> (5-aza-dC)-mediated induction of Foxp3 expression in CD8 + T cells in vitro . Naive CD4 + and CD8 + T cells (both Foxp3 GFP− CD25 − CD62L high ) were FACS-isolated to high purity from pooled lymph nodes and spleen of adult Foxp3 GFP mice and subjected to T cell receptor stimulation in vitro (anti-CD3/CD28-coated beads) in the presence of IL-2 (100 U/ml), either alone or with added TGF-β (0.5 ng/ml) and/or 5-aza-dC (0.5 µM), as indicated. Cultures were analyzed at day 3 for Foxp3 GFP and CD25 expression among gated CD4 + or CD8 + T cells. (A) Representative flow cytometry of Foxp3 GFP /CD25 expression among gated CD4 + (top, black) and CD8 + (bottom, blue) T cells. Numbers in dot plots indicate the percentages of cells within the respective quadrant. (B,C) Median fluorescence intensities (MFIs) of CD25 expression among gated (B) CD4 + T cells (left: Foxp3 GFP− , right: Foxp3 GFP+ ) and (C) CD8 + T cells (left: Foxp3 GFP− , right: Foxp3 GFP+ ). Graphs show mean percentages ±SD of CD25 MFI from triplicate wells. The level of significance was determined by two-way ANOVA with Tukey’s multiple comparison test: **** P ≤ 0.0001 and P > 0.05 (ns).
    Meyer Dc, supplied by SCHNEEBERGER, used in various techniques. Bioz Stars score: 89/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    4Gene weinstein dc
    <t>5-Aza-2′-deoxycytidine</t> (5-aza-dC)-mediated induction of Foxp3 expression in CD8 + T cells in vitro . Naive CD4 + and CD8 + T cells (both Foxp3 GFP− CD25 − CD62L high ) were FACS-isolated to high purity from pooled lymph nodes and spleen of adult Foxp3 GFP mice and subjected to T cell receptor stimulation in vitro (anti-CD3/CD28-coated beads) in the presence of IL-2 (100 U/ml), either alone or with added TGF-β (0.5 ng/ml) and/or 5-aza-dC (0.5 µM), as indicated. Cultures were analyzed at day 3 for Foxp3 GFP and CD25 expression among gated CD4 + or CD8 + T cells. (A) Representative flow cytometry of Foxp3 GFP /CD25 expression among gated CD4 + (top, black) and CD8 + (bottom, blue) T cells. Numbers in dot plots indicate the percentages of cells within the respective quadrant. (B,C) Median fluorescence intensities (MFIs) of CD25 expression among gated (B) CD4 + T cells (left: Foxp3 GFP− , right: Foxp3 GFP+ ) and (C) CD8 + T cells (left: Foxp3 GFP− , right: Foxp3 GFP+ ). Graphs show mean percentages ±SD of CD25 MFI from triplicate wells. The level of significance was determined by two-way ANOVA with Tukey’s multiple comparison test: **** P ≤ 0.0001 and P > 0.05 (ns).
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    91
    Janssen valla dc
    <t>5-Aza-2′-deoxycytidine</t> (5-aza-dC)-mediated induction of Foxp3 expression in CD8 + T cells in vitro . Naive CD4 + and CD8 + T cells (both Foxp3 GFP− CD25 − CD62L high ) were FACS-isolated to high purity from pooled lymph nodes and spleen of adult Foxp3 GFP mice and subjected to T cell receptor stimulation in vitro (anti-CD3/CD28-coated beads) in the presence of IL-2 (100 U/ml), either alone or with added TGF-β (0.5 ng/ml) and/or 5-aza-dC (0.5 µM), as indicated. Cultures were analyzed at day 3 for Foxp3 GFP and CD25 expression among gated CD4 + or CD8 + T cells. (A) Representative flow cytometry of Foxp3 GFP /CD25 expression among gated CD4 + (top, black) and CD8 + (bottom, blue) T cells. Numbers in dot plots indicate the percentages of cells within the respective quadrant. (B,C) Median fluorescence intensities (MFIs) of CD25 expression among gated (B) CD4 + T cells (left: Foxp3 GFP− , right: Foxp3 GFP+ ) and (C) CD8 + T cells (left: Foxp3 GFP− , right: Foxp3 GFP+ ). Graphs show mean percentages ±SD of CD25 MFI from triplicate wells. The level of significance was determined by two-way ANOVA with Tukey’s multiple comparison test: **** P ≤ 0.0001 and P > 0.05 (ns).
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    Image Search Results


    Differences in sialylation between parental, non-invasive LNCaP and bone metastatic C4-2B cells ( A ) Flow cytometric determination of increased cell surface expression levels of α2,3- (left panel) and equal expression levels of α2,6- (right panel) linked sialic acid residues in C4-2B cells, using the lectins MAA and SNA, respectively. Single cell suspensions of LNCaP and C4-2B cells were incubated with biotinylated-MAA and SNA, followed by Fluorescein Avidin DCS and analysed on the Cell Lab Quanta SC MPL, stainings without the particular lectins were used as controls. Each experiment was performed at least three times. ( B ) Lectin blot analysis for total expression levels of α2,3- and α2,6-linked sialic acids in LNCaP and C4-2B cell lysates, containing 25 μg protein. Lysates were analysed by 4–20% gradient or 7.5% SDS/PAGE, and blotted with MAA (left panel) and SNA (right panel). Sialidase treatment (0.5 U/ml sodium citrate buffer at pH 6) of the blotted membranes for 16 h at 37°C, serves as control for the presence of sialic acid residues. Scion Image densitometry analysis of bands indicating the presence of α2,3- and α2,6-linked sialic acids in both cell lines (lower panels) and evaluation of the increased density at 125 kDa in C4-2B cells (side left panel) ( C ) Transcriptional expression levels of ST genes resulting in α2,3- and α2,6-linked sialic acid residues. Expression levels were determined by QPCR, normalized against HPRT (hypoxanthine–guanine phosphoribosyltransferase) and levels present in C4-2B cells are expressed relative as compared with the expression in the parental, non-invasive LNCaP cells. Analysed and evaluated data are means±S.D. from at least three independent experiments, asterisks indicate statistical difference from parental, non-invasive LNCaP control cells ( P

    Journal: Bioscience Reports

    Article Title: Carbohydrate-to-carbohydrate interactions between α2,3-linked sialic acids on α2 integrin subunits and asialo-GM1 underlie the bone metastatic behaviour of LNCAP-derivative C4-2B prostate cancer cells

    doi: 10.1042/BSR20140096

    Figure Lengend Snippet: Differences in sialylation between parental, non-invasive LNCaP and bone metastatic C4-2B cells ( A ) Flow cytometric determination of increased cell surface expression levels of α2,3- (left panel) and equal expression levels of α2,6- (right panel) linked sialic acid residues in C4-2B cells, using the lectins MAA and SNA, respectively. Single cell suspensions of LNCaP and C4-2B cells were incubated with biotinylated-MAA and SNA, followed by Fluorescein Avidin DCS and analysed on the Cell Lab Quanta SC MPL, stainings without the particular lectins were used as controls. Each experiment was performed at least three times. ( B ) Lectin blot analysis for total expression levels of α2,3- and α2,6-linked sialic acids in LNCaP and C4-2B cell lysates, containing 25 μg protein. Lysates were analysed by 4–20% gradient or 7.5% SDS/PAGE, and blotted with MAA (left panel) and SNA (right panel). Sialidase treatment (0.5 U/ml sodium citrate buffer at pH 6) of the blotted membranes for 16 h at 37°C, serves as control for the presence of sialic acid residues. Scion Image densitometry analysis of bands indicating the presence of α2,3- and α2,6-linked sialic acids in both cell lines (lower panels) and evaluation of the increased density at 125 kDa in C4-2B cells (side left panel) ( C ) Transcriptional expression levels of ST genes resulting in α2,3- and α2,6-linked sialic acid residues. Expression levels were determined by QPCR, normalized against HPRT (hypoxanthine–guanine phosphoribosyltransferase) and levels present in C4-2B cells are expressed relative as compared with the expression in the parental, non-invasive LNCaP cells. Analysed and evaluated data are means±S.D. from at least three independent experiments, asterisks indicate statistical difference from parental, non-invasive LNCaP control cells ( P

    Article Snippet: Biotinylated-MAA (Maackia amurensis agglutinin) and SNA (Sambucus nigra agglutinin), as well as fluorescein-labelled SNA, Fluorescein Avidin DCS and Vectashield mounting medium were obtained from Vector Laboratories.

    Techniques: Flow Cytometry, Expressing, Incubation, Avidin-Biotin Assay, SDS Page, Real-time Polymerase Chain Reaction

    Effect of morphine on HCV NS5 protein expression in Huh.8 ( A ) and FCA-1 ( B ) cells. Huh.8 and FCA-1 cells (10 5 cells/well) plated in a 24-well plate were incubated with or without (Control) morphine at 10 −6 mol/L for 72 hours. Cell lysates were quantified by DC protein assay kit. Equal amounts (0.5 μg) of protein extracted from treated and untreated Huh.8 and FCA-1 cells were applied onto a NC membrane for immunoblot assay. The results were recorded on the film (2 minutes exposure). One representative result from three experiments is shown.

    Journal: The American Journal of Pathology

    Article Title: Morphine Enhances Hepatitis C Virus (HCV) Replicon Expression

    doi:

    Figure Lengend Snippet: Effect of morphine on HCV NS5 protein expression in Huh.8 ( A ) and FCA-1 ( B ) cells. Huh.8 and FCA-1 cells (10 5 cells/well) plated in a 24-well plate were incubated with or without (Control) morphine at 10 −6 mol/L for 72 hours. Cell lysates were quantified by DC protein assay kit. Equal amounts (0.5 μg) of protein extracted from treated and untreated Huh.8 and FCA-1 cells were applied onto a NC membrane for immunoblot assay. The results were recorded on the film (2 minutes exposure). One representative result from three experiments is shown.

    Article Snippet: The protein concentration was determined by detergent-compatible (DC) protein assay kit (Bio-Rad, Hercules, CA).

    Techniques: Expressing, Incubation, DC Protein Assay

    5-Aza-2′-deoxycytidine (5-aza-dC)-mediated induction of Foxp3 expression in CD8 + T cells in vitro . Naive CD4 + and CD8 + T cells (both Foxp3 GFP− CD25 − CD62L high ) were FACS-isolated to high purity from pooled lymph nodes and spleen of adult Foxp3 GFP mice and subjected to T cell receptor stimulation in vitro (anti-CD3/CD28-coated beads) in the presence of IL-2 (100 U/ml), either alone or with added TGF-β (0.5 ng/ml) and/or 5-aza-dC (0.5 µM), as indicated. Cultures were analyzed at day 3 for Foxp3 GFP and CD25 expression among gated CD4 + or CD8 + T cells. (A) Representative flow cytometry of Foxp3 GFP /CD25 expression among gated CD4 + (top, black) and CD8 + (bottom, blue) T cells. Numbers in dot plots indicate the percentages of cells within the respective quadrant. (B,C) Median fluorescence intensities (MFIs) of CD25 expression among gated (B) CD4 + T cells (left: Foxp3 GFP− , right: Foxp3 GFP+ ) and (C) CD8 + T cells (left: Foxp3 GFP− , right: Foxp3 GFP+ ). Graphs show mean percentages ±SD of CD25 MFI from triplicate wells. The level of significance was determined by two-way ANOVA with Tukey’s multiple comparison test: **** P ≤ 0.0001 and P > 0.05 (ns).

    Journal: Frontiers in Immunology

    Article Title: Critical Role of TGF-β and IL-2 Receptor Signaling in Foxp3 Induction by an Inhibitor of DNA Methylation

    doi: 10.3389/fimmu.2018.00125

    Figure Lengend Snippet: 5-Aza-2′-deoxycytidine (5-aza-dC)-mediated induction of Foxp3 expression in CD8 + T cells in vitro . Naive CD4 + and CD8 + T cells (both Foxp3 GFP− CD25 − CD62L high ) were FACS-isolated to high purity from pooled lymph nodes and spleen of adult Foxp3 GFP mice and subjected to T cell receptor stimulation in vitro (anti-CD3/CD28-coated beads) in the presence of IL-2 (100 U/ml), either alone or with added TGF-β (0.5 ng/ml) and/or 5-aza-dC (0.5 µM), as indicated. Cultures were analyzed at day 3 for Foxp3 GFP and CD25 expression among gated CD4 + or CD8 + T cells. (A) Representative flow cytometry of Foxp3 GFP /CD25 expression among gated CD4 + (top, black) and CD8 + (bottom, blue) T cells. Numbers in dot plots indicate the percentages of cells within the respective quadrant. (B,C) Median fluorescence intensities (MFIs) of CD25 expression among gated (B) CD4 + T cells (left: Foxp3 GFP− , right: Foxp3 GFP+ ) and (C) CD8 + T cells (left: Foxp3 GFP− , right: Foxp3 GFP+ ). Graphs show mean percentages ±SD of CD25 MFI from triplicate wells. The level of significance was determined by two-way ANOVA with Tukey’s multiple comparison test: **** P ≤ 0.0001 and P > 0.05 (ns).

    Article Snippet: Importantly, in the absence of added TGF-β, 40 µM SB431542 efficiently inhibited 5-aza-dC-mediated Foxp3 induction (Figures D,E), even at high concentrations of 5-aza-dC.

    Techniques: Expressing, In Vitro, FACS, Isolation, Mouse Assay, Flow Cytometry, Cytometry, Fluorescence

    Impact of FCS-/T cell-derived TGF-β on 5-aza-2′-deoxycytidine (5-aza-dC)-mediated Foxp3 + iTreg cell generation, in the absence of exogenously added TGF-β. CD4 + Foxp3 GFP− T cell stimulation cultures (for experimental details, see Figure 1 ) were either left untreated or supplemented with added TGF-β (0.5 ng/ml) or titrating amounts of 5-aza-dC (0.1, 0.25, 0.5, or 1.0 µM), with or without neutralizing anti-TGF-β mAbs (20 µg/ml), as indicated. Cultures were analyzed at day 3 for Foxp3 GFP and CD25 expression among gated CD4 + T cells. Note that Figure S1 in Supplementary Material depicts experimental details on anti-TGF-β mAb titration. (A) 20 µg/ml anti-TGF-β mAb efficiently abrogates Foxp3 induction in T cell receptor stimulation cultures without (left) or with (right) exogenously added TGF-β (0.5 ng/ml). (B–D) Inhibition of 5-aza-dC-driven Foxp3 + iTreg cell generation by anti-TGF-β mAb-mediated TGF-β blockage, in the absence of added TGF-β. (B) Representative flow cytometry of Foxp3 GFP /CD25 expression among gated CD4 + T cells, and (C) composite percentages of CD4 + Foxp3 GFP+ iTreg cells at indicated culture conditions. (D) Percentage inhibition of 5-aza-dC-mediated Foxp3 GFP+ iTreg cell generation by TGF-β blockage (see Materials and Methods for details). Symbols and horizontal lines (A,C) indicate triplicate wells and mean values, respectively. Numbers in dot plots (B) indicate the percentages of cells within the respective quadrant. Data are representative of at least two independent experiments.

    Journal: Frontiers in Immunology

    Article Title: Critical Role of TGF-β and IL-2 Receptor Signaling in Foxp3 Induction by an Inhibitor of DNA Methylation

    doi: 10.3389/fimmu.2018.00125

    Figure Lengend Snippet: Impact of FCS-/T cell-derived TGF-β on 5-aza-2′-deoxycytidine (5-aza-dC)-mediated Foxp3 + iTreg cell generation, in the absence of exogenously added TGF-β. CD4 + Foxp3 GFP− T cell stimulation cultures (for experimental details, see Figure 1 ) were either left untreated or supplemented with added TGF-β (0.5 ng/ml) or titrating amounts of 5-aza-dC (0.1, 0.25, 0.5, or 1.0 µM), with or without neutralizing anti-TGF-β mAbs (20 µg/ml), as indicated. Cultures were analyzed at day 3 for Foxp3 GFP and CD25 expression among gated CD4 + T cells. Note that Figure S1 in Supplementary Material depicts experimental details on anti-TGF-β mAb titration. (A) 20 µg/ml anti-TGF-β mAb efficiently abrogates Foxp3 induction in T cell receptor stimulation cultures without (left) or with (right) exogenously added TGF-β (0.5 ng/ml). (B–D) Inhibition of 5-aza-dC-driven Foxp3 + iTreg cell generation by anti-TGF-β mAb-mediated TGF-β blockage, in the absence of added TGF-β. (B) Representative flow cytometry of Foxp3 GFP /CD25 expression among gated CD4 + T cells, and (C) composite percentages of CD4 + Foxp3 GFP+ iTreg cells at indicated culture conditions. (D) Percentage inhibition of 5-aza-dC-mediated Foxp3 GFP+ iTreg cell generation by TGF-β blockage (see Materials and Methods for details). Symbols and horizontal lines (A,C) indicate triplicate wells and mean values, respectively. Numbers in dot plots (B) indicate the percentages of cells within the respective quadrant. Data are representative of at least two independent experiments.

    Article Snippet: Importantly, in the absence of added TGF-β, 40 µM SB431542 efficiently inhibited 5-aza-dC-mediated Foxp3 induction (Figures D,E), even at high concentrations of 5-aza-dC.

    Techniques: Derivative Assay, Cell Stimulation, Expressing, Titration, Inhibition, Flow Cytometry, Cytometry

    Impact of TGF-βRII signaling on 5-aza-2′-deoxycytidine (5-aza-dC)-mediated Foxp3 + iTreg cell generation. See Figure 1 for experimental details on naive CD4 + Foxp3 GFP− T cell isolation. T cell receptor (TCR) stimulation cultures were analyzed on day 3 for Foxp3 GFP and CD25 expression among gated CD4 + T cells. (A–C) Genetic abrogation of TGF-βR signaling. In the absence of added TGF-β, CD4 + Foxp3 GFP− T cells expressing a wild-type (WT) TGF-βRII or a dominant-negative TGF-βRII (dnTGF-βRII) were TCR stimulated in the absence or presence of titrating amounts of 5-aza-dC (0.1, 0.25, 0.5, or 1.0 µM). (A) Representative flow cytometry and (B) composite percentages of CD4 + Foxp3 GFP+ iTreg cells at indicated culture conditions. (C) Percentage inhibition of 5-aza-dC-mediated Foxp3 GFP+ iTreg cell generation by dnTGF-βRII expression (see Materials and Methods for details). (D–F) Pharmacological abrogation of TGF-βR signaling. TGF-βR signaling-proficient, naive CD4 + Foxp3 GFP− T cells were subjected to TCR stimulation, either alone or with TGF-β (0.5 ng/ml) or titrating amounts of 5-aza-dC (0.1, 0.25, 0.5, or 1.0 µM), in the absence and presence of 40 µM SB431542, as indicated. Note that Figure S2 in Supplementary Material depicts experimental details on the titration of SB431542. (D) Representative flow cytometry and (E) composite percentages of CD4 + Foxp3 GFP+ iTreg cells at indicated culture conditions. (F) Percentage inhibition of 5-aza-dC-mediated Foxp3 GFP+ iTreg cell generation by SB431542 (see Materials and Methods for details). Numbers in dot plots (A,D) indicate the percentages of cells within the respective quadrant. Symbols and horizontal lines (B,E) indicate triplicate wells and mean values, respectively. (B,E) The level of significance was determined by multiple t -tests (unpaired). * P ≤ 0.05. Data are representative of at least two independent experiments.

    Journal: Frontiers in Immunology

    Article Title: Critical Role of TGF-β and IL-2 Receptor Signaling in Foxp3 Induction by an Inhibitor of DNA Methylation

    doi: 10.3389/fimmu.2018.00125

    Figure Lengend Snippet: Impact of TGF-βRII signaling on 5-aza-2′-deoxycytidine (5-aza-dC)-mediated Foxp3 + iTreg cell generation. See Figure 1 for experimental details on naive CD4 + Foxp3 GFP− T cell isolation. T cell receptor (TCR) stimulation cultures were analyzed on day 3 for Foxp3 GFP and CD25 expression among gated CD4 + T cells. (A–C) Genetic abrogation of TGF-βR signaling. In the absence of added TGF-β, CD4 + Foxp3 GFP− T cells expressing a wild-type (WT) TGF-βRII or a dominant-negative TGF-βRII (dnTGF-βRII) were TCR stimulated in the absence or presence of titrating amounts of 5-aza-dC (0.1, 0.25, 0.5, or 1.0 µM). (A) Representative flow cytometry and (B) composite percentages of CD4 + Foxp3 GFP+ iTreg cells at indicated culture conditions. (C) Percentage inhibition of 5-aza-dC-mediated Foxp3 GFP+ iTreg cell generation by dnTGF-βRII expression (see Materials and Methods for details). (D–F) Pharmacological abrogation of TGF-βR signaling. TGF-βR signaling-proficient, naive CD4 + Foxp3 GFP− T cells were subjected to TCR stimulation, either alone or with TGF-β (0.5 ng/ml) or titrating amounts of 5-aza-dC (0.1, 0.25, 0.5, or 1.0 µM), in the absence and presence of 40 µM SB431542, as indicated. Note that Figure S2 in Supplementary Material depicts experimental details on the titration of SB431542. (D) Representative flow cytometry and (E) composite percentages of CD4 + Foxp3 GFP+ iTreg cells at indicated culture conditions. (F) Percentage inhibition of 5-aza-dC-mediated Foxp3 GFP+ iTreg cell generation by SB431542 (see Materials and Methods for details). Numbers in dot plots (A,D) indicate the percentages of cells within the respective quadrant. Symbols and horizontal lines (B,E) indicate triplicate wells and mean values, respectively. (B,E) The level of significance was determined by multiple t -tests (unpaired). * P ≤ 0.05. Data are representative of at least two independent experiments.

    Article Snippet: Importantly, in the absence of added TGF-β, 40 µM SB431542 efficiently inhibited 5-aza-dC-mediated Foxp3 induction (Figures D,E), even at high concentrations of 5-aza-dC.

    Techniques: Cell Isolation, Expressing, Dominant Negative Mutation, Flow Cytometry, Cytometry, Inhibition, Titration

    Impact of IL-2 and IL-2R signaling on 5-Aza-2′-deoxycytidine (5-aza-dC)-mediated Foxp3 + iTreg cell generation. Naive CD4 + Foxp3 GFP− T cells were T cell receptor stimulated with anti-CD3/CD28-coated beads either alone (shown in gray) or with added TGF-β and 5-aza-dC (0.5 ng/ml and 0.5 µM; shown in green), added TGF-β (0.5 ng/ml; shown in blue) or 5-aza-dC (0.5 µM; shown in red). As indicated, either IL-2 (100 U/ml) or neutralizing anti-IL-2 mAb (10 µg/ml) were added. Cultures were analyzed on day 3 for Foxp3 GFP and CD25 expression among gated CD4 + T cells. (A) Representative histograms of Foxp3 GFP expression among gated CD4 + T cells and (B) composite percentages of CD4 + Foxp3 + iTreg cells at indicated culture conditions. (C) Representative histograms and (D) median fluorescence intensities (MFIs) of CD25 expression among gated CD4 + T cells. (E) Percentage inhibition/enhancement of TGF-β-mediated (blue bars) and 5-aza-dC-mediated (red bars) Foxp3 GFP+ iTreg cell generation by the addition of exogenous IL-2 (left) or anti-IL-2 mAbs (right), as indicated (see Materials and Methods for details). Numbers in histograms show (A) the mean values of the percentage of Foxp3 GFP+ cells and (C) MFI values of CD25 expression ±SD at indicated culture conditions. Graphs (B,D) depict mean ± SD. Data are representative of two independent experiments.

    Journal: Frontiers in Immunology

    Article Title: Critical Role of TGF-β and IL-2 Receptor Signaling in Foxp3 Induction by an Inhibitor of DNA Methylation

    doi: 10.3389/fimmu.2018.00125

    Figure Lengend Snippet: Impact of IL-2 and IL-2R signaling on 5-Aza-2′-deoxycytidine (5-aza-dC)-mediated Foxp3 + iTreg cell generation. Naive CD4 + Foxp3 GFP− T cells were T cell receptor stimulated with anti-CD3/CD28-coated beads either alone (shown in gray) or with added TGF-β and 5-aza-dC (0.5 ng/ml and 0.5 µM; shown in green), added TGF-β (0.5 ng/ml; shown in blue) or 5-aza-dC (0.5 µM; shown in red). As indicated, either IL-2 (100 U/ml) or neutralizing anti-IL-2 mAb (10 µg/ml) were added. Cultures were analyzed on day 3 for Foxp3 GFP and CD25 expression among gated CD4 + T cells. (A) Representative histograms of Foxp3 GFP expression among gated CD4 + T cells and (B) composite percentages of CD4 + Foxp3 + iTreg cells at indicated culture conditions. (C) Representative histograms and (D) median fluorescence intensities (MFIs) of CD25 expression among gated CD4 + T cells. (E) Percentage inhibition/enhancement of TGF-β-mediated (blue bars) and 5-aza-dC-mediated (red bars) Foxp3 GFP+ iTreg cell generation by the addition of exogenous IL-2 (left) or anti-IL-2 mAbs (right), as indicated (see Materials and Methods for details). Numbers in histograms show (A) the mean values of the percentage of Foxp3 GFP+ cells and (C) MFI values of CD25 expression ±SD at indicated culture conditions. Graphs (B,D) depict mean ± SD. Data are representative of two independent experiments.

    Article Snippet: Importantly, in the absence of added TGF-β, 40 µM SB431542 efficiently inhibited 5-aza-dC-mediated Foxp3 induction (Figures D,E), even at high concentrations of 5-aza-dC.

    Techniques: Expressing, Fluorescence, Inhibition

    5-Aza-2′-deoxycytidine (5-aza-dC) promotes IL-2R signaling during Foxp3 + iTreg cell generation by enhancing CD25 and pSTAT5 expression. Naive CD4 + Foxp3 GFP− T cells were T cell receptor stimulated (see Figure 1 for experimental details) either alone or in the presence of TGF-β (0.5 ng/ml) or 5-aza-dC (0.5 µM). (A–C) Kinetics of CD25 expression. (A) Representative flow cytometry of CD25 expression among gated total CD4 + T cells at 24 h, (B) CD4 + Foxp3 GFP− and CD4 + Foxp3 GFP+ T cells at 72 h after initiation of cultures, and (C) corresponding median fluorescence intensities (MFIs) of CD25 expression (left: 24 h, CD4 + total; right: 72 h, CD4 + Foxp3 GFP− and CD4 + Foxp3 GFP+ ). (D–F) STAT5 phosphorylation. (D) Representative flow cytometry of pSTAT5 expression among gated total CD4 + T cells at 24 h and (E) CD4 + Foxp3 GFP− and CD4 + Foxp3 GFP+ T cells at 72 h after initiation of cultures, and (C) corresponding MFIs of pSTAT5 expression (left: 24 h, CD4 + total; right: 72 h, CD4 + Foxp3 GFP− and CD4 + Foxp3 GFP+ ). Numbers in histograms and graphs show mean percentages ± SD of (A–C) CD25 and (D–F) pSTAT5 MFIs from triplicate wells [not detectable (n.d.)]. (C,F) The level of significance was determined by two-way ANOVA with Tukey’s multiple comparison test: **** P ≤ 0.0001, *** P ≤ 0.001, and P > 0.05 (ns). Data are representative of two independent experiments.

    Journal: Frontiers in Immunology

    Article Title: Critical Role of TGF-β and IL-2 Receptor Signaling in Foxp3 Induction by an Inhibitor of DNA Methylation

    doi: 10.3389/fimmu.2018.00125

    Figure Lengend Snippet: 5-Aza-2′-deoxycytidine (5-aza-dC) promotes IL-2R signaling during Foxp3 + iTreg cell generation by enhancing CD25 and pSTAT5 expression. Naive CD4 + Foxp3 GFP− T cells were T cell receptor stimulated (see Figure 1 for experimental details) either alone or in the presence of TGF-β (0.5 ng/ml) or 5-aza-dC (0.5 µM). (A–C) Kinetics of CD25 expression. (A) Representative flow cytometry of CD25 expression among gated total CD4 + T cells at 24 h, (B) CD4 + Foxp3 GFP− and CD4 + Foxp3 GFP+ T cells at 72 h after initiation of cultures, and (C) corresponding median fluorescence intensities (MFIs) of CD25 expression (left: 24 h, CD4 + total; right: 72 h, CD4 + Foxp3 GFP− and CD4 + Foxp3 GFP+ ). (D–F) STAT5 phosphorylation. (D) Representative flow cytometry of pSTAT5 expression among gated total CD4 + T cells at 24 h and (E) CD4 + Foxp3 GFP− and CD4 + Foxp3 GFP+ T cells at 72 h after initiation of cultures, and (C) corresponding MFIs of pSTAT5 expression (left: 24 h, CD4 + total; right: 72 h, CD4 + Foxp3 GFP− and CD4 + Foxp3 GFP+ ). Numbers in histograms and graphs show mean percentages ± SD of (A–C) CD25 and (D–F) pSTAT5 MFIs from triplicate wells [not detectable (n.d.)]. (C,F) The level of significance was determined by two-way ANOVA with Tukey’s multiple comparison test: **** P ≤ 0.0001, *** P ≤ 0.001, and P > 0.05 (ns). Data are representative of two independent experiments.

    Article Snippet: Importantly, in the absence of added TGF-β, 40 µM SB431542 efficiently inhibited 5-aza-dC-mediated Foxp3 induction (Figures D,E), even at high concentrations of 5-aza-dC.

    Techniques: Expressing, Flow Cytometry, Cytometry, Fluorescence

    Interplay of 5-aza-2′-deoxycytidine (5-aza-dC) with IL-2R/TGF-βR signaling during Foxp3 + iTreg cell generation. CD4 + Foxp3 GFP− T cells with a naive phenotype (CD25 − CD62L high ) were FACS-isolated to high purity (99.7%) from pooled lymph nodes and spleen of adult Foxp3 GFP mice and subjected to T cell receptor (TCR) stimulation in vitro (anti-CD3/CD28-coated beads), either alone or with TGF-β and/or 5-aza-dC, as indicated. Note that all cultures were performed in the absence of exogenous IL-2. At indicated time points, cultures were analyzed for Foxp3 GFP and CD25 expression among gated CD4 + T cells. (A–D) 5-aza-dC synergizes with TGF-β in promoting Foxp3 + CD25 + iTreg cell generation. As indicated, TCR stimulation cultures were either left untreated or supplemented with TGF-β (5 ng/ml) and/or titrating amounts of 5-aza-dC (0.1, 0.25, 0.5, or 1.0 µM). (A) Representative flow cytometry of Foxp3 GFP /CD25 expression among gated CD4 + T cells, and (B) composite percentages of CD4 + Foxp3 GFP+ iTreg cells at day 3 of cultures. (C) Corresponding representative flow cytometry of cell viability (FSC/SSC), and (D) composite percentages of viable cells within the indicated FSC/SSC gate. The level of significance was determined by multiple t -tests (unpaired). P ≤ 0.05 (*). (E–H) Naive CD4 + Foxp3 GFP− T cell stimulation cultures were either left untreated (gray circles) or supplemented with 0.5 ng/ml TGF-β (blue triangles) or 0.5 µM 5-aza-dC (red circles) and subjected to further analysis at indicated time points. (E) Kinetics of Foxp3 mRNA expression. (F,G) Kinetics of Foxp3 protein expression. (F) Composite percentages and (G) representative histograms of Foxp3 GFP expression among gated CD4 + T cells at indicated time points and culture conditions. (H) Kinetics of Smad7 mRNA expression. Gene expression (E,H) was determined by real-time RT-PCR employing FACS-purified populations of total viable cells (i.e., irrespective of their Foxp3 GFP expression status). HPRT1 was used for normalization. Where indicated, the level of significance was determined by two-way ANOVA with Tukey’s multiple comparison test: **** P ≤ 0.0001 and P > 0.05 (ns). Numbers (A,C,G) show the percentage of gated cells within the respective quadrant or gate. Symbols and horizontal lines (B,D) indicate triplicate wells and mean values, respectively. Data are representative of at least two independent experiments.

    Journal: Frontiers in Immunology

    Article Title: Critical Role of TGF-β and IL-2 Receptor Signaling in Foxp3 Induction by an Inhibitor of DNA Methylation

    doi: 10.3389/fimmu.2018.00125

    Figure Lengend Snippet: Interplay of 5-aza-2′-deoxycytidine (5-aza-dC) with IL-2R/TGF-βR signaling during Foxp3 + iTreg cell generation. CD4 + Foxp3 GFP− T cells with a naive phenotype (CD25 − CD62L high ) were FACS-isolated to high purity (99.7%) from pooled lymph nodes and spleen of adult Foxp3 GFP mice and subjected to T cell receptor (TCR) stimulation in vitro (anti-CD3/CD28-coated beads), either alone or with TGF-β and/or 5-aza-dC, as indicated. Note that all cultures were performed in the absence of exogenous IL-2. At indicated time points, cultures were analyzed for Foxp3 GFP and CD25 expression among gated CD4 + T cells. (A–D) 5-aza-dC synergizes with TGF-β in promoting Foxp3 + CD25 + iTreg cell generation. As indicated, TCR stimulation cultures were either left untreated or supplemented with TGF-β (5 ng/ml) and/or titrating amounts of 5-aza-dC (0.1, 0.25, 0.5, or 1.0 µM). (A) Representative flow cytometry of Foxp3 GFP /CD25 expression among gated CD4 + T cells, and (B) composite percentages of CD4 + Foxp3 GFP+ iTreg cells at day 3 of cultures. (C) Corresponding representative flow cytometry of cell viability (FSC/SSC), and (D) composite percentages of viable cells within the indicated FSC/SSC gate. The level of significance was determined by multiple t -tests (unpaired). P ≤ 0.05 (*). (E–H) Naive CD4 + Foxp3 GFP− T cell stimulation cultures were either left untreated (gray circles) or supplemented with 0.5 ng/ml TGF-β (blue triangles) or 0.5 µM 5-aza-dC (red circles) and subjected to further analysis at indicated time points. (E) Kinetics of Foxp3 mRNA expression. (F,G) Kinetics of Foxp3 protein expression. (F) Composite percentages and (G) representative histograms of Foxp3 GFP expression among gated CD4 + T cells at indicated time points and culture conditions. (H) Kinetics of Smad7 mRNA expression. Gene expression (E,H) was determined by real-time RT-PCR employing FACS-purified populations of total viable cells (i.e., irrespective of their Foxp3 GFP expression status). HPRT1 was used for normalization. Where indicated, the level of significance was determined by two-way ANOVA with Tukey’s multiple comparison test: **** P ≤ 0.0001 and P > 0.05 (ns). Numbers (A,C,G) show the percentage of gated cells within the respective quadrant or gate. Symbols and horizontal lines (B,D) indicate triplicate wells and mean values, respectively. Data are representative of at least two independent experiments.

    Article Snippet: Importantly, in the absence of added TGF-β, 40 µM SB431542 efficiently inhibited 5-aza-dC-mediated Foxp3 induction (Figures D,E), even at high concentrations of 5-aza-dC.

    Techniques: FACS, Isolation, Mouse Assay, In Vitro, Expressing, Flow Cytometry, Cytometry, Cell Stimulation, Quantitative RT-PCR, Purification