reagents flow cytometry antibodies anti mouse cd4 magnet beads  (Becton Dickinson)

 
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    Structured Review

    Becton Dickinson reagents flow cytometry antibodies anti mouse cd4 magnet beads
    MLN DC and LPDC take up and present CBir1 flagellin to T cells after mucosal challenge in vivo . Wild-type and TCRβ×δ −/− mice were gavaged with Alexa 647-labeled CBir1 flagellin. Uptake of CBir1 flagellin by MLN DC and LPDC was determined 2 hrs later by flow <t>cytometry</t> ( A ). When cultured with <t>CD4</t> T cells of CBir1 Tg mice, LPDC from CBir1-gavaged TCRβxδ KO, but not wild-type, mice stimulated T cell proliferation ( B ). Data are reflective of 3 independent experiments. *p
    Reagents Flow Cytometry Antibodies Anti Mouse Cd4 Magnet Beads, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/reagents flow cytometry antibodies anti mouse cd4 magnet beads/product/Becton Dickinson
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    Images

    1) Product Images from "TLR5 mediates CD172α+ intestinal lamina propria dendritic cell induction of Th17 cells"

    Article Title: TLR5 mediates CD172α+ intestinal lamina propria dendritic cell induction of Th17 cells

    Journal: Scientific Reports

    doi: 10.1038/srep22040

    MLN DC and LPDC take up and present CBir1 flagellin to T cells after mucosal challenge in vivo . Wild-type and TCRβ×δ −/− mice were gavaged with Alexa 647-labeled CBir1 flagellin. Uptake of CBir1 flagellin by MLN DC and LPDC was determined 2 hrs later by flow cytometry ( A ). When cultured with CD4 T cells of CBir1 Tg mice, LPDC from CBir1-gavaged TCRβxδ KO, but not wild-type, mice stimulated T cell proliferation ( B ). Data are reflective of 3 independent experiments. *p
    Figure Legend Snippet: MLN DC and LPDC take up and present CBir1 flagellin to T cells after mucosal challenge in vivo . Wild-type and TCRβ×δ −/− mice were gavaged with Alexa 647-labeled CBir1 flagellin. Uptake of CBir1 flagellin by MLN DC and LPDC was determined 2 hrs later by flow cytometry ( A ). When cultured with CD4 T cells of CBir1 Tg mice, LPDC from CBir1-gavaged TCRβxδ KO, but not wild-type, mice stimulated T cell proliferation ( B ). Data are reflective of 3 independent experiments. *p

    Techniques Used: In Vivo, Mouse Assay, Labeling, Flow Cytometry, Cytometry, Cell Culture

    More LPDCs express TLR5 than spleen DCs and most CD172α + LPDCs are also TLR5 + . Spleen cells and LP cells were isolated from C57BL/6 mice. After depleting CD4 + T cells, CD4 − cells were stained with CD11c, CD11b, CD103, and TLR5 and analyzed by flow cytometry. ( A ) CD11c + cell expression of TLR5 was shown. ( B ) TLR5 expression by CD11b + and CD11b − DCs by gating on CD11c + population. ( C ) CD11c + DC expression of CD103 and CD11b by gating on CD11c + population. ( D ) Expression of TLR5 and CD172α by CD103 + DC and CD103 − DCs in LP of wild-type C57BL/6 mice by gating on CD11c + CD11b + population. ( E,F ) CD172α expression by CD103 + DC and CD103 − DCs in LP of wild-type ( E ) and TLR5 KO C57BL/6 mice ( F ) by gating on CD11c + population. FACS plots are reflective of 2–4 independent experiments.
    Figure Legend Snippet: More LPDCs express TLR5 than spleen DCs and most CD172α + LPDCs are also TLR5 + . Spleen cells and LP cells were isolated from C57BL/6 mice. After depleting CD4 + T cells, CD4 − cells were stained with CD11c, CD11b, CD103, and TLR5 and analyzed by flow cytometry. ( A ) CD11c + cell expression of TLR5 was shown. ( B ) TLR5 expression by CD11b + and CD11b − DCs by gating on CD11c + population. ( C ) CD11c + DC expression of CD103 and CD11b by gating on CD11c + population. ( D ) Expression of TLR5 and CD172α by CD103 + DC and CD103 − DCs in LP of wild-type C57BL/6 mice by gating on CD11c + CD11b + population. ( E,F ) CD172α expression by CD103 + DC and CD103 − DCs in LP of wild-type ( E ) and TLR5 KO C57BL/6 mice ( F ) by gating on CD11c + population. FACS plots are reflective of 2–4 independent experiments.

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

    Related Articles

    Flow Cytometry:

    Article Title: TLR5 mediates CD172α+ intestinal lamina propria dendritic cell induction of Th17 cells
    Article Snippet: .. Antibodies and reagents Flow cytometry antibodies anti-mouse CD4-magnet beads were purchased from BD Bioscience. .. Anti-CD4, anti-IL-17, anti-IFNγ, anti-CD11c, anti-CD11b, anti-CD103, anti-CD172α were purchased from Biolegends.

    Cytometry:

    Article Title: TLR5 mediates CD172α+ intestinal lamina propria dendritic cell induction of Th17 cells
    Article Snippet: .. Antibodies and reagents Flow cytometry antibodies anti-mouse CD4-magnet beads were purchased from BD Bioscience. .. Anti-CD4, anti-IL-17, anti-IFNγ, anti-CD11c, anti-CD11b, anti-CD103, anti-CD172α were purchased from Biolegends.

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    Becton Dickinson anti cd4 magnetic beads
    Surface phenotype of pulmonary T cells in CBA/J and C57BL/6 mice. C57BL/6 and CBA/J mice were infected with an aerosolized dose of Mtb and at various timepoints post-infection lungs were removed and processed for flow cytometry. Absolute numbers of IFN-γ + <t>CD4</t> + ( a ) or CD8 + ( b ) T cells after 4 hr ex vivo stimulation with anti-CD3/CD28/GolgiSTOP, with representative flow plots at day 150 post-infection. Absolute numbers of CD8 + T cells expressing CD69 ( c ), Tim3 ( d ), or PD-1 ( e ) after Mtb infection. ( f ) Absolute number of CD8 + T cells expressing both PD-1 and CD122. Data representative of at least two independent experiments with 4 mice per group per timepoint. * p
    Anti Cd4 Magnetic Beads, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti mouse cd4 magnetic beads
    Dose response of TGFβ on expression of SOCS3, RORγt, Foxp3, and Th17 cell development in naïve <t>CD4</t> + T Cells (A) B6 CD4 + CD25 − T cells were cultured with increasing doses of TGFβ (0.01 to 10 ng/ml) in the absence or presence of IL-6 (20 ng/ml) for 2 h, and total RNA analyzed by RT-PCR to measure SOCS3 and GAPDH mRNA levels. mRNA levels in the untreated sample were set at 1.0, and results are expressed as fold induction over these control levels. (B) B6 CD4 + CD25 − T cells were cultured with anti-CD3/CD28 and anti-IFNγ (10 μg/ml), anti-IL-4 (10 μg/ml), and increasing doses of TGF-β in the absence or presence of IL-6 (20 ng/ml). RORγt expression was measured by real-time PCR 48 h after culture. The data were normalized to β2 microglobulin as a reference. The experiments were repeated three times with consistent results. (C) To measure IL-17 production, five days after culture, the cells were restimulated with PMA/ionomycin for 5 h and intracellular IL-17 was measured by flow cytometry. (D) Foxp3 expression was determined by flow cytometry five days after culture. Representative of three experiments.
    Anti Mouse Cd4 Magnetic Beads, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson cd4
    TGF-β-mediated inhibition of naïve T cell proliferation is comparable between wildtype and Drak2-/- T cells. A) <t>CD4</t> + CD25 - CD44 lo naïve cells were purified from OT-II and OT-II . Drak2-/- mice and stimulated with irradiated splenocytes loaded with 10μM OVA 323 peptide in the presence or absence of 10-fold TGF-β titrations for three days. The number of live, divided Foxp3 - CD4 + cells are shown for each titration. Cells were obtained from one OT-II or OT-II . Drak2-/- mouse and tested in quadruplicate. Data are representative of five separate experiments. B) CD8 + CD25 - CD44 lo CD62L hi naïve cells were purified from OT-I and OT-I . Drak2-/- mice and stimulated with splenocytes loaded with 100pM OVA 257 peptide in the presence or absence of 10-fold TGF-β titrations. Two days later, cells were harvested and analyzed by flow cytometry. The number of live, divided CD8 + cells are shown for each titration. Cells were obtained from one OT-I or OT-I . Drak2-/- mouse and tested in quadruplicate. Data are representative of three separate experiments. There was no significant difference in the response of the wildtype and Drak2-/- cells according to the Mann-Whitney U -test.
    Cd4, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 1465 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti cd4 antibody coated magnetic beads
    Cytokine production by <t>CD4</t> + cells isolated from tumor tissue. Tumor tissues were collected from mice (N=3–6 per group, two independent experiments). The CD4 + cells were isolated from tumor tissue using anti-CD4 antibody-coated magnetic beads. Cells were cultured for 48 h with Con A. The concentrations of IFN-γ, IL-4 and IL-10 in cell culture supernatants were measured by ELISA. Differences by mean ± SD were estimated. The statistical significance was calculated (for details see Fig. 3 ).
    Anti Cd4 Antibody Coated Magnetic Beads, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Surface phenotype of pulmonary T cells in CBA/J and C57BL/6 mice. C57BL/6 and CBA/J mice were infected with an aerosolized dose of Mtb and at various timepoints post-infection lungs were removed and processed for flow cytometry. Absolute numbers of IFN-γ + CD4 + ( a ) or CD8 + ( b ) T cells after 4 hr ex vivo stimulation with anti-CD3/CD28/GolgiSTOP, with representative flow plots at day 150 post-infection. Absolute numbers of CD8 + T cells expressing CD69 ( c ), Tim3 ( d ), or PD-1 ( e ) after Mtb infection. ( f ) Absolute number of CD8 + T cells expressing both PD-1 and CD122. Data representative of at least two independent experiments with 4 mice per group per timepoint. * p

    Journal: PLoS ONE

    Article Title: Clonal Expansions of CD8+ T Cells with IL-10 Secreting Capacity Occur during Chronic Mycobacterium tuberculosis Infection

    doi: 10.1371/journal.pone.0058612

    Figure Lengend Snippet: Surface phenotype of pulmonary T cells in CBA/J and C57BL/6 mice. C57BL/6 and CBA/J mice were infected with an aerosolized dose of Mtb and at various timepoints post-infection lungs were removed and processed for flow cytometry. Absolute numbers of IFN-γ + CD4 + ( a ) or CD8 + ( b ) T cells after 4 hr ex vivo stimulation with anti-CD3/CD28/GolgiSTOP, with representative flow plots at day 150 post-infection. Absolute numbers of CD8 + T cells expressing CD69 ( c ), Tim3 ( d ), or PD-1 ( e ) after Mtb infection. ( f ) Absolute number of CD8 + T cells expressing both PD-1 and CD122. Data representative of at least two independent experiments with 4 mice per group per timepoint. * p

    Article Snippet: CD8+ Vβ8+ T cells were obtained from the CD4neg fraction after treatment with anti-CD4 magnetic beads (BD), then stained with PE anti-Vβ8 (eBioscience) and purified using anti-PE magnetic particles (BD).

    Techniques: Crocin Bleaching Assay, Mouse Assay, Infection, Flow Cytometry, Cytometry, Ex Vivo, Expressing

    Vβ TcR expression in CBA/J and C57BL/6 mice. C57BL/6 and CBA/J mice were infected with an aerosol dose of Mtb and at various timepoints post-infection lungs were removed and processed for flow cytometry. Percentages of CD8 + ( a ) or CD4 + ( b ) T cells expressing specific Vβ TcRs at day 120 post-infection. Percentages of CD8 + T cells expressing Vβ8 ( c ) or Vβ14 ( d ) TcR over the course of Mtb infection. ( e ) Percentages of Vβ8 + or Vβ14 + CD69 + CD8 + pulmonary T cells over time. ( f ) Vβ8 + and Vβ8 neg T cells from day 120 post-infection were cultured for 72 hr with anti-CD3/CD28 and IL-10 SFU determined by ELISpot. ( g ) Supernatants from (a) were analyzed for IFN-γ levels by ELISA. Data representative of at least two independent experiments with 4 mice per group per timepoint. * p

    Journal: PLoS ONE

    Article Title: Clonal Expansions of CD8+ T Cells with IL-10 Secreting Capacity Occur during Chronic Mycobacterium tuberculosis Infection

    doi: 10.1371/journal.pone.0058612

    Figure Lengend Snippet: Vβ TcR expression in CBA/J and C57BL/6 mice. C57BL/6 and CBA/J mice were infected with an aerosol dose of Mtb and at various timepoints post-infection lungs were removed and processed for flow cytometry. Percentages of CD8 + ( a ) or CD4 + ( b ) T cells expressing specific Vβ TcRs at day 120 post-infection. Percentages of CD8 + T cells expressing Vβ8 ( c ) or Vβ14 ( d ) TcR over the course of Mtb infection. ( e ) Percentages of Vβ8 + or Vβ14 + CD69 + CD8 + pulmonary T cells over time. ( f ) Vβ8 + and Vβ8 neg T cells from day 120 post-infection were cultured for 72 hr with anti-CD3/CD28 and IL-10 SFU determined by ELISpot. ( g ) Supernatants from (a) were analyzed for IFN-γ levels by ELISA. Data representative of at least two independent experiments with 4 mice per group per timepoint. * p

    Article Snippet: CD8+ Vβ8+ T cells were obtained from the CD4neg fraction after treatment with anti-CD4 magnetic beads (BD), then stained with PE anti-Vβ8 (eBioscience) and purified using anti-PE magnetic particles (BD).

    Techniques: Expressing, Crocin Bleaching Assay, Mouse Assay, Infection, Flow Cytometry, Cytometry, Cell Culture, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay

    CD8 + T cell depletion in CBA/J mice. CBA/J mice were infected with an aerosolized dose of Mtb and from day 90–120 were treated weekly with depletion antibody via intraperitoneal injection then sacrificed at day 125 post-infection. ( a ) Lungs of anti-CD8 + T cell depleted or control mice were homogenized and plated on 7H11 plates and CFU enumerated after 21 days. Absolute number of total CD4 + T cells ( b ) or IFN-γ + CD4 + T cells ( c ) after CD8 + T cell depletion as determined by flow cytometry. ( d ) Levels of pulmonary IL-10 after CD8 + T cell depletion as determined by ELISA. ( e ) Lungs of anti-Vβ8 depleted or control mice were homogenized and plated onto 7H11 plates and CFU enumerated. Control group = no treatment and isotype control. Results representative of at least two independent experiments with 5–10 mice per group. Depletion resulted in 95% reduction in cell number, as determined by flow cytometry. * p

    Journal: PLoS ONE

    Article Title: Clonal Expansions of CD8+ T Cells with IL-10 Secreting Capacity Occur during Chronic Mycobacterium tuberculosis Infection

    doi: 10.1371/journal.pone.0058612

    Figure Lengend Snippet: CD8 + T cell depletion in CBA/J mice. CBA/J mice were infected with an aerosolized dose of Mtb and from day 90–120 were treated weekly with depletion antibody via intraperitoneal injection then sacrificed at day 125 post-infection. ( a ) Lungs of anti-CD8 + T cell depleted or control mice were homogenized and plated on 7H11 plates and CFU enumerated after 21 days. Absolute number of total CD4 + T cells ( b ) or IFN-γ + CD4 + T cells ( c ) after CD8 + T cell depletion as determined by flow cytometry. ( d ) Levels of pulmonary IL-10 after CD8 + T cell depletion as determined by ELISA. ( e ) Lungs of anti-Vβ8 depleted or control mice were homogenized and plated onto 7H11 plates and CFU enumerated. Control group = no treatment and isotype control. Results representative of at least two independent experiments with 5–10 mice per group. Depletion resulted in 95% reduction in cell number, as determined by flow cytometry. * p

    Article Snippet: CD8+ Vβ8+ T cells were obtained from the CD4neg fraction after treatment with anti-CD4 magnetic beads (BD), then stained with PE anti-Vβ8 (eBioscience) and purified using anti-PE magnetic particles (BD).

    Techniques: Crocin Bleaching Assay, Mouse Assay, Infection, Injection, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    Accumulation and characterization of CBA/J CD8 + T cells. C57BL/6, CBA/J, and CBA/J IL-10 −/− mice were infected with an aerosolized dose of Mtb , and at various times post-infection lungs were removed. ( a, b ) CBA/J and C57BL/6 lung cell were analyzed by flow cytometry for CD4 + and CD8 + T cells. ( c ) Ratio of CBA/J CD4 + to CD8 + T cells representative of four independent experiments with 5 mice per group, per timepoint. ( d ) C57BL/6 and CBA/J lungs were homogenized and plated on 7H11 plates for CFU enumeration. ( e, f ) 24 hr prior to necropsy mice were injected with BrdU, and lung cells were analyzed for expression of BrdU + CD4 + or CD8 + T cells. ( g, h ) Absolute numbers of CD4 + or CD8 + T cells in wild-type or IL-10 −/− CBA/J mice as determined by flow cytometry. Results representative of at least three independent experiments with 5 mice per group, per timepoint. * p

    Journal: PLoS ONE

    Article Title: Clonal Expansions of CD8+ T Cells with IL-10 Secreting Capacity Occur during Chronic Mycobacterium tuberculosis Infection

    doi: 10.1371/journal.pone.0058612

    Figure Lengend Snippet: Accumulation and characterization of CBA/J CD8 + T cells. C57BL/6, CBA/J, and CBA/J IL-10 −/− mice were infected with an aerosolized dose of Mtb , and at various times post-infection lungs were removed. ( a, b ) CBA/J and C57BL/6 lung cell were analyzed by flow cytometry for CD4 + and CD8 + T cells. ( c ) Ratio of CBA/J CD4 + to CD8 + T cells representative of four independent experiments with 5 mice per group, per timepoint. ( d ) C57BL/6 and CBA/J lungs were homogenized and plated on 7H11 plates for CFU enumeration. ( e, f ) 24 hr prior to necropsy mice were injected with BrdU, and lung cells were analyzed for expression of BrdU + CD4 + or CD8 + T cells. ( g, h ) Absolute numbers of CD4 + or CD8 + T cells in wild-type or IL-10 −/− CBA/J mice as determined by flow cytometry. Results representative of at least three independent experiments with 5 mice per group, per timepoint. * p

    Article Snippet: CD8+ Vβ8+ T cells were obtained from the CD4neg fraction after treatment with anti-CD4 magnetic beads (BD), then stained with PE anti-Vβ8 (eBioscience) and purified using anti-PE magnetic particles (BD).

    Techniques: Crocin Bleaching Assay, Mouse Assay, Infection, Flow Cytometry, Cytometry, Injection, Expressing

    Dose response of TGFβ on expression of SOCS3, RORγt, Foxp3, and Th17 cell development in naïve CD4 + T Cells (A) B6 CD4 + CD25 − T cells were cultured with increasing doses of TGFβ (0.01 to 10 ng/ml) in the absence or presence of IL-6 (20 ng/ml) for 2 h, and total RNA analyzed by RT-PCR to measure SOCS3 and GAPDH mRNA levels. mRNA levels in the untreated sample were set at 1.0, and results are expressed as fold induction over these control levels. (B) B6 CD4 + CD25 − T cells were cultured with anti-CD3/CD28 and anti-IFNγ (10 μg/ml), anti-IL-4 (10 μg/ml), and increasing doses of TGF-β in the absence or presence of IL-6 (20 ng/ml). RORγt expression was measured by real-time PCR 48 h after culture. The data were normalized to β2 microglobulin as a reference. The experiments were repeated three times with consistent results. (C) To measure IL-17 production, five days after culture, the cells were restimulated with PMA/ionomycin for 5 h and intracellular IL-17 was measured by flow cytometry. (D) Foxp3 expression was determined by flow cytometry five days after culture. Representative of three experiments.

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

    Article Title: TGF-? promotes Th17 cell development through inhibition of SOCS3

    doi: 10.4049/jimmunol.0801986

    Figure Lengend Snippet: Dose response of TGFβ on expression of SOCS3, RORγt, Foxp3, and Th17 cell development in naïve CD4 + T Cells (A) B6 CD4 + CD25 − T cells were cultured with increasing doses of TGFβ (0.01 to 10 ng/ml) in the absence or presence of IL-6 (20 ng/ml) for 2 h, and total RNA analyzed by RT-PCR to measure SOCS3 and GAPDH mRNA levels. mRNA levels in the untreated sample were set at 1.0, and results are expressed as fold induction over these control levels. (B) B6 CD4 + CD25 − T cells were cultured with anti-CD3/CD28 and anti-IFNγ (10 μg/ml), anti-IL-4 (10 μg/ml), and increasing doses of TGF-β in the absence or presence of IL-6 (20 ng/ml). RORγt expression was measured by real-time PCR 48 h after culture. The data were normalized to β2 microglobulin as a reference. The experiments were repeated three times with consistent results. (C) To measure IL-17 production, five days after culture, the cells were restimulated with PMA/ionomycin for 5 h and intracellular IL-17 was measured by flow cytometry. (D) Foxp3 expression was determined by flow cytometry five days after culture. Representative of three experiments.

    Article Snippet: CD4+ T cells were isolated using anti-mouse CD4-magnetic beads (BD Biosiences, San Diego, CA).

    Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry

    Effect of SOCS3 knockdown on RORγt and Th17 cell development Naïve CD4 + CD25 − T cells were transfected with 100 nM of control or SOCS3 siRNAs using the Dharma FECT ™ 1 reagent and Mouse T Cell Nucleofector Kit. Twenty-four h after transfection, T cells were stimulated with anti-CD3/CD28 mAbs in the presence of IL-6 (20 ng/ml), TGF-β (5 ng/ml), or both. (A) SOCS-3 expression was measured with 1 h of IL-6 (20 ng/ml) stimulation by real-time PCR. The data were normalized to β2 microglobulin reference. * P ≤ 0.05. (B) RORγt expression was measured 48 h later by real-time PCR. The data were normalized to β2 microglobulin reference. * P ≤ 0.05. (C) IL-17 and IFNγ production was determined 5 days later by flow cytometry. (D) Foxp3 expression was determined 5 days later by flow cytometry. Representative of three experiments.

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

    Article Title: TGF-? promotes Th17 cell development through inhibition of SOCS3

    doi: 10.4049/jimmunol.0801986

    Figure Lengend Snippet: Effect of SOCS3 knockdown on RORγt and Th17 cell development Naïve CD4 + CD25 − T cells were transfected with 100 nM of control or SOCS3 siRNAs using the Dharma FECT ™ 1 reagent and Mouse T Cell Nucleofector Kit. Twenty-four h after transfection, T cells were stimulated with anti-CD3/CD28 mAbs in the presence of IL-6 (20 ng/ml), TGF-β (5 ng/ml), or both. (A) SOCS-3 expression was measured with 1 h of IL-6 (20 ng/ml) stimulation by real-time PCR. The data were normalized to β2 microglobulin reference. * P ≤ 0.05. (B) RORγt expression was measured 48 h later by real-time PCR. The data were normalized to β2 microglobulin reference. * P ≤ 0.05. (C) IL-17 and IFNγ production was determined 5 days later by flow cytometry. (D) Foxp3 expression was determined 5 days later by flow cytometry. Representative of three experiments.

    Article Snippet: CD4+ T cells were isolated using anti-mouse CD4-magnetic beads (BD Biosiences, San Diego, CA).

    Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry

    Less Th17 cells in lamina propria of TGFβRII DN mice and lower RORγt expression of TGFβRII DN CD4 + T cells stimulated with IL-6 and TGF-β (A) Lamina propria CD4 + T cells were isolated from wild type or TGF-βRII DN B6 mice and stimulated with PMA/ionomycin for 5 h. Intracellular IL-17 and IFNγ production was measured by flow cytometry . (B) Splenic CD4 + T cells were isolated from wild type or TGFβRII DN B6 mice and cultured with anti-CD3/CD28 in the absence or presence of IL-6 (20 ng/ml), anti-IFNγ (10 mg/ml), anti-IL-4 (10 mg/ml), and TGF-β (5 ng/ml). RNA was isolated and RORγt expression was analyzed using real-time PCR. Expression of β-microglobulin was used as house-keeping gene control. Representative of three experiments. * P ≤ 0.05. (C) WT or TGF-β RIIDN CD4 + T cells were treated with medium alone (UN) or IL-6 (20 ng/ml) for up to 2 h, and total RNA analyzed by RT-PCR to measure SOCS3 and GAPDH mRNA levels. mRNA levels in the untreated sample were set at 1.0, and results are expressed as fold induction over these control levels. (D) To evaluate STAT3 activation, WT or TGF-β RIIDN CD4 + T cells were incubated with IL-6 (20 ng/ml), TGF-β (5 ng/ml), or IL-6 plus TGF-β for 1 h, then cell lysates immunoblotted with anti-phospho-Tyr-STAT3. The membranes were stripped and reprobed with anti-STAT3 and anti-actin as a loading control. STAT3 phosphorylation level in the untreated sample was set at 1.0, and results are calculated as fold activation over these control levels. The data shown are representative of at least three experiments.

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

    Article Title: TGF-? promotes Th17 cell development through inhibition of SOCS3

    doi: 10.4049/jimmunol.0801986

    Figure Lengend Snippet: Less Th17 cells in lamina propria of TGFβRII DN mice and lower RORγt expression of TGFβRII DN CD4 + T cells stimulated with IL-6 and TGF-β (A) Lamina propria CD4 + T cells were isolated from wild type or TGF-βRII DN B6 mice and stimulated with PMA/ionomycin for 5 h. Intracellular IL-17 and IFNγ production was measured by flow cytometry . (B) Splenic CD4 + T cells were isolated from wild type or TGFβRII DN B6 mice and cultured with anti-CD3/CD28 in the absence or presence of IL-6 (20 ng/ml), anti-IFNγ (10 mg/ml), anti-IL-4 (10 mg/ml), and TGF-β (5 ng/ml). RNA was isolated and RORγt expression was analyzed using real-time PCR. Expression of β-microglobulin was used as house-keeping gene control. Representative of three experiments. * P ≤ 0.05. (C) WT or TGF-β RIIDN CD4 + T cells were treated with medium alone (UN) or IL-6 (20 ng/ml) for up to 2 h, and total RNA analyzed by RT-PCR to measure SOCS3 and GAPDH mRNA levels. mRNA levels in the untreated sample were set at 1.0, and results are expressed as fold induction over these control levels. (D) To evaluate STAT3 activation, WT or TGF-β RIIDN CD4 + T cells were incubated with IL-6 (20 ng/ml), TGF-β (5 ng/ml), or IL-6 plus TGF-β for 1 h, then cell lysates immunoblotted with anti-phospho-Tyr-STAT3. The membranes were stripped and reprobed with anti-STAT3 and anti-actin as a loading control. STAT3 phosphorylation level in the untreated sample was set at 1.0, and results are calculated as fold activation over these control levels. The data shown are representative of at least three experiments.

    Article Snippet: CD4+ T cells were isolated using anti-mouse CD4-magnetic beads (BD Biosiences, San Diego, CA).

    Techniques: Mouse Assay, Expressing, Isolation, Flow Cytometry, Cytometry, Cell Culture, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Activation Assay, Incubation

    IL-21- and TGF-β-induced SOCS3 expression and STAT3 activation in CD4 + T cells (A) CD4 + T cells were treated with medium (UN), IL-21 (10 ng/ml), or IL-21 plus TGF-β (5 ng/ml) for up to 1 h, and total RNA analyzed by RPA to measure SOCS-3, and GAPDH mRNA levels. mRNA levels in the untreated sample were set at 1.0, and results are expressed as fold induction over these control levels. (B) To evaluate STAT3 activation, CD4 + T cells were incubated with IL-21 (10 ng/ml), TGF-β (5 ng/ml) or IL-21 plus TGF-β for up to 4 h, then cell lysates immunoblotted with anti-phospho-Tyr-STAT3 or anti-phospho-Ser-STAT3. The membranes were stripped and reprobed with anti-STAT3 and anti-actin as loading controls. STAT3 phosphorylation level in the untreated sample was set at 1.0, and results are calculated as fold activation over these control levels. The data shown are representative of at least three experiments.

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

    Article Title: TGF-? promotes Th17 cell development through inhibition of SOCS3

    doi: 10.4049/jimmunol.0801986

    Figure Lengend Snippet: IL-21- and TGF-β-induced SOCS3 expression and STAT3 activation in CD4 + T cells (A) CD4 + T cells were treated with medium (UN), IL-21 (10 ng/ml), or IL-21 plus TGF-β (5 ng/ml) for up to 1 h, and total RNA analyzed by RPA to measure SOCS-3, and GAPDH mRNA levels. mRNA levels in the untreated sample were set at 1.0, and results are expressed as fold induction over these control levels. (B) To evaluate STAT3 activation, CD4 + T cells were incubated with IL-21 (10 ng/ml), TGF-β (5 ng/ml) or IL-21 plus TGF-β for up to 4 h, then cell lysates immunoblotted with anti-phospho-Tyr-STAT3 or anti-phospho-Ser-STAT3. The membranes were stripped and reprobed with anti-STAT3 and anti-actin as loading controls. STAT3 phosphorylation level in the untreated sample was set at 1.0, and results are calculated as fold activation over these control levels. The data shown are representative of at least three experiments.

    Article Snippet: CD4+ T cells were isolated using anti-mouse CD4-magnetic beads (BD Biosiences, San Diego, CA).

    Techniques: Expressing, Activation Assay, Recombinase Polymerase Amplification, Incubation

    Effect of IL-6 and TGF-β1 on Th17 cell development, expression of SOCS3 and RORγt, and activation of STAT3 in CD4 + T Cells (A) B6 CD4 + T cells were cultured with anti-CD3/CD28 in the absence or presence of IL-6 (20 ng/ml), anti-IFNγ (10 μg/ml), anti-IL-12(10 μg/ml), anti-IL-4 (10 μg/ml), and TGF-β (5 ng/ml). Seven days later, the cells were restimulated with PMA/ionomycin for 5 h and intracellular IL-17 and IFNγ production was measured by flow cytometry. (B) To measure IL-6- and TGF-β induction of SOCS3, CD4 + T cells were treated with medium (UN), IL-6 (20 ng/ml), or IL-6 plus TGF-β (5 ng/ml) for up to 2 h, and total RNA analyzed by RT-PCR to measure SOCS3, and GAPDH mRNA levels. mRNA levels in the untreated sample were set at 1.0, and results are expressed as fold induction over these control levels. (C) To determine the effect of TGF-β on SOCS3 promoter activity, CD4 + T cells were transfected with a 1619-bp (−1492 to +127 bp) murine SOCS-3 promoter, and treated with IL-6 (20 ng/ml), TGF-β (5 ng/ml), or both for 12 h, and the luciferase activity of each sample was normalized to the total protein concentration in each well. Luciferase activity from the untreated sample was arbitrarily set at 1 for calculation of fold induction. * P ≤ 0.05. (D) To evaluate STAT3 activation, CD4 + T cells were incubated with IL-6 (20 ng/ml), or IL-6 plus TGF-β (5 ng/ml) for up to 8 h, then cell lysates were immunoblotted with anti-phospho-Tyr-STAT3 or anti-phospho-Ser-STAT3. The membranes were stripped and reprobed with anti-STAT3 and anti-actin as a loading control. The data shown are representative of at least three experiments. STAT3 phosphorylation level in the untreated sample was set at 1.0, and results are calculated as fold activation over these control levels. ( E ) RORγt expression of B6 CD4 + CD25 − T cells cultured with anti-CD3/CD28, anti-IFNγ, and anti-IL-4 in the absence or presence of IL-6 (20 ng/ml) and TGF-β (10 ng/ml) was determined by real-time PCR 48 h after culture. The data were normalized to a β2 microglobulin reference. The experiments were repeated three times with consistent results. * P ≤ 0.05.

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

    Article Title: TGF-? promotes Th17 cell development through inhibition of SOCS3

    doi: 10.4049/jimmunol.0801986

    Figure Lengend Snippet: Effect of IL-6 and TGF-β1 on Th17 cell development, expression of SOCS3 and RORγt, and activation of STAT3 in CD4 + T Cells (A) B6 CD4 + T cells were cultured with anti-CD3/CD28 in the absence or presence of IL-6 (20 ng/ml), anti-IFNγ (10 μg/ml), anti-IL-12(10 μg/ml), anti-IL-4 (10 μg/ml), and TGF-β (5 ng/ml). Seven days later, the cells were restimulated with PMA/ionomycin for 5 h and intracellular IL-17 and IFNγ production was measured by flow cytometry. (B) To measure IL-6- and TGF-β induction of SOCS3, CD4 + T cells were treated with medium (UN), IL-6 (20 ng/ml), or IL-6 plus TGF-β (5 ng/ml) for up to 2 h, and total RNA analyzed by RT-PCR to measure SOCS3, and GAPDH mRNA levels. mRNA levels in the untreated sample were set at 1.0, and results are expressed as fold induction over these control levels. (C) To determine the effect of TGF-β on SOCS3 promoter activity, CD4 + T cells were transfected with a 1619-bp (−1492 to +127 bp) murine SOCS-3 promoter, and treated with IL-6 (20 ng/ml), TGF-β (5 ng/ml), or both for 12 h, and the luciferase activity of each sample was normalized to the total protein concentration in each well. Luciferase activity from the untreated sample was arbitrarily set at 1 for calculation of fold induction. * P ≤ 0.05. (D) To evaluate STAT3 activation, CD4 + T cells were incubated with IL-6 (20 ng/ml), or IL-6 plus TGF-β (5 ng/ml) for up to 8 h, then cell lysates were immunoblotted with anti-phospho-Tyr-STAT3 or anti-phospho-Ser-STAT3. The membranes were stripped and reprobed with anti-STAT3 and anti-actin as a loading control. The data shown are representative of at least three experiments. STAT3 phosphorylation level in the untreated sample was set at 1.0, and results are calculated as fold activation over these control levels. ( E ) RORγt expression of B6 CD4 + CD25 − T cells cultured with anti-CD3/CD28, anti-IFNγ, and anti-IL-4 in the absence or presence of IL-6 (20 ng/ml) and TGF-β (10 ng/ml) was determined by real-time PCR 48 h after culture. The data were normalized to a β2 microglobulin reference. The experiments were repeated three times with consistent results. * P ≤ 0.05.

    Article Snippet: CD4+ T cells were isolated using anti-mouse CD4-magnetic beads (BD Biosiences, San Diego, CA).

    Techniques: Expressing, Activation Assay, Cell Culture, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Transfection, Luciferase, Protein Concentration, Incubation, Real-time Polymerase Chain Reaction

    TGF-β-mediated inhibition of naïve T cell proliferation is comparable between wildtype and Drak2-/- T cells. A) CD4 + CD25 - CD44 lo naïve cells were purified from OT-II and OT-II . Drak2-/- mice and stimulated with irradiated splenocytes loaded with 10μM OVA 323 peptide in the presence or absence of 10-fold TGF-β titrations for three days. The number of live, divided Foxp3 - CD4 + cells are shown for each titration. Cells were obtained from one OT-II or OT-II . Drak2-/- mouse and tested in quadruplicate. Data are representative of five separate experiments. B) CD8 + CD25 - CD44 lo CD62L hi naïve cells were purified from OT-I and OT-I . Drak2-/- mice and stimulated with splenocytes loaded with 100pM OVA 257 peptide in the presence or absence of 10-fold TGF-β titrations. Two days later, cells were harvested and analyzed by flow cytometry. The number of live, divided CD8 + cells are shown for each titration. Cells were obtained from one OT-I or OT-I . Drak2-/- mouse and tested in quadruplicate. Data are representative of three separate experiments. There was no significant difference in the response of the wildtype and Drak2-/- cells according to the Mann-Whitney U -test.

    Journal: PLoS ONE

    Article Title: Drak2 Does Not Regulate TGF-β Signaling in T Cells

    doi: 10.1371/journal.pone.0123650

    Figure Lengend Snippet: TGF-β-mediated inhibition of naïve T cell proliferation is comparable between wildtype and Drak2-/- T cells. A) CD4 + CD25 - CD44 lo naïve cells were purified from OT-II and OT-II . Drak2-/- mice and stimulated with irradiated splenocytes loaded with 10μM OVA 323 peptide in the presence or absence of 10-fold TGF-β titrations for three days. The number of live, divided Foxp3 - CD4 + cells are shown for each titration. Cells were obtained from one OT-II or OT-II . Drak2-/- mouse and tested in quadruplicate. Data are representative of five separate experiments. B) CD8 + CD25 - CD44 lo CD62L hi naïve cells were purified from OT-I and OT-I . Drak2-/- mice and stimulated with splenocytes loaded with 100pM OVA 257 peptide in the presence or absence of 10-fold TGF-β titrations. Two days later, cells were harvested and analyzed by flow cytometry. The number of live, divided CD8 + cells are shown for each titration. Cells were obtained from one OT-I or OT-I . Drak2-/- mouse and tested in quadruplicate. Data are representative of three separate experiments. There was no significant difference in the response of the wildtype and Drak2-/- cells according to the Mann-Whitney U -test.

    Article Snippet: For analysis of phosphorylated Smad2/3, cells were stained with antibodies specific for CD4 and CD8, then fixed with 1X BD Phosflow Lyse/Fix Buffer and permeabilized with BD Phosflow Perm Buffer III according to manufacturer’s instructions (BD Biosciences) and stained with anti-pSmad2/3 antibody (BD Biosciences).

    Techniques: Inhibition, Purification, Mouse Assay, Irradiation, Titration, Flow Cytometry, Cytometry, MANN-WHITNEY

    Enhanced susceptibility to death of Drak2-/- T cells compared to wildtype T cells is independent of TGF-β signaling in vitro . A) CD4 + CD25 - CD44 lo or B) CD8 + CD25 - CD44 lo naïve cells were purified from wildtype, Drak2-/- , DNRII , and DNRII . Drak2-/- mice and stimulated with anti-CD3 and anti-CD28 for 2–3 days. The percent of nonviable CD4 + or CD8 + T cells is shown. Cells were obtained from one mouse per group and tested in quadruplicate. Data are representative of four separate experiments. **P

    Journal: PLoS ONE

    Article Title: Drak2 Does Not Regulate TGF-β Signaling in T Cells

    doi: 10.1371/journal.pone.0123650

    Figure Lengend Snippet: Enhanced susceptibility to death of Drak2-/- T cells compared to wildtype T cells is independent of TGF-β signaling in vitro . A) CD4 + CD25 - CD44 lo or B) CD8 + CD25 - CD44 lo naïve cells were purified from wildtype, Drak2-/- , DNRII , and DNRII . Drak2-/- mice and stimulated with anti-CD3 and anti-CD28 for 2–3 days. The percent of nonviable CD4 + or CD8 + T cells is shown. Cells were obtained from one mouse per group and tested in quadruplicate. Data are representative of four separate experiments. **P

    Article Snippet: For analysis of phosphorylated Smad2/3, cells were stained with antibodies specific for CD4 and CD8, then fixed with 1X BD Phosflow Lyse/Fix Buffer and permeabilized with BD Phosflow Perm Buffer III according to manufacturer’s instructions (BD Biosciences) and stained with anti-pSmad2/3 antibody (BD Biosciences).

    Techniques: In Vitro, Purification, Mouse Assay

    Smad2 and Smad2/3 complex phosphorylation is not enhanced in Drak2-/- T cells compared to wildtype T cells. A) Wildtype and Drak2-/- splenocytes, and FACS sorted naïve CD4 + and CD8 + T cells were stimulated for 2 hours with anti-CD3 and anti-CD28, with or without 2 ng/ml TGF-β for one additional hour. Cells were lysed and analyzed by western blot with antibodies specific for Smad2, phosphorylated Smad2, and HSP90 as a loading control. Cells were pooled from 9 wildtype and 8 Drak2-/- mice. Data are representative of two independent experiments. B) Wildtype and Drak2-/- splenocytes were stimulated for 2 hours with anti-CD3 and anti-CD28 with or without increasing concentrations of TGF-β for one additional hour. The cells were harvested, stained with antibodies specific for CD4, CD8, and pSmad2/3, and analyzed by flow cytometry. The average mean fluorescence intensity (MFI) of pSmad2/3 expression is shown for 3 mice per group. There was no significant difference in the response of the wildtype and Drak2-/- cells according to the Mann-Whitney U -test. Data are representative of 3 independent experiments.

    Journal: PLoS ONE

    Article Title: Drak2 Does Not Regulate TGF-β Signaling in T Cells

    doi: 10.1371/journal.pone.0123650

    Figure Lengend Snippet: Smad2 and Smad2/3 complex phosphorylation is not enhanced in Drak2-/- T cells compared to wildtype T cells. A) Wildtype and Drak2-/- splenocytes, and FACS sorted naïve CD4 + and CD8 + T cells were stimulated for 2 hours with anti-CD3 and anti-CD28, with or without 2 ng/ml TGF-β for one additional hour. Cells were lysed and analyzed by western blot with antibodies specific for Smad2, phosphorylated Smad2, and HSP90 as a loading control. Cells were pooled from 9 wildtype and 8 Drak2-/- mice. Data are representative of two independent experiments. B) Wildtype and Drak2-/- splenocytes were stimulated for 2 hours with anti-CD3 and anti-CD28 with or without increasing concentrations of TGF-β for one additional hour. The cells were harvested, stained with antibodies specific for CD4, CD8, and pSmad2/3, and analyzed by flow cytometry. The average mean fluorescence intensity (MFI) of pSmad2/3 expression is shown for 3 mice per group. There was no significant difference in the response of the wildtype and Drak2-/- cells according to the Mann-Whitney U -test. Data are representative of 3 independent experiments.

    Article Snippet: For analysis of phosphorylated Smad2/3, cells were stained with antibodies specific for CD4 and CD8, then fixed with 1X BD Phosflow Lyse/Fix Buffer and permeabilized with BD Phosflow Perm Buffer III according to manufacturer’s instructions (BD Biosciences) and stained with anti-pSmad2/3 antibody (BD Biosciences).

    Techniques: FACS, Western Blot, Mouse Assay, Staining, Flow Cytometry, Cytometry, Fluorescence, Expressing, MANN-WHITNEY

    Smad2 translocation is not enhanced in Drak2-/- T cells compared to wildtype T cells. Wildtype and Drak2-/- CD4 + cells were negatively selected with Miltenyi magnetic beads and stimulated on anti-CD3-coated coverglass slides along with soluble anti-CD28 for 24 hours. Half of the cells were treated with TGF-β for the final 20 minutes of culture. Cells were fixed, permeabilized, and stained with DAPI, Phalloidin, and anti-Smad2. Images were collected via confocal microscopy. n = 2 mice per group. Data are representative of two independent experiments.

    Journal: PLoS ONE

    Article Title: Drak2 Does Not Regulate TGF-β Signaling in T Cells

    doi: 10.1371/journal.pone.0123650

    Figure Lengend Snippet: Smad2 translocation is not enhanced in Drak2-/- T cells compared to wildtype T cells. Wildtype and Drak2-/- CD4 + cells were negatively selected with Miltenyi magnetic beads and stimulated on anti-CD3-coated coverglass slides along with soluble anti-CD28 for 24 hours. Half of the cells were treated with TGF-β for the final 20 minutes of culture. Cells were fixed, permeabilized, and stained with DAPI, Phalloidin, and anti-Smad2. Images were collected via confocal microscopy. n = 2 mice per group. Data are representative of two independent experiments.

    Article Snippet: For analysis of phosphorylated Smad2/3, cells were stained with antibodies specific for CD4 and CD8, then fixed with 1X BD Phosflow Lyse/Fix Buffer and permeabilized with BD Phosflow Perm Buffer III according to manufacturer’s instructions (BD Biosciences) and stained with anti-pSmad2/3 antibody (BD Biosciences).

    Techniques: Translocation Assay, Magnetic Beads, Staining, Confocal Microscopy, Mouse Assay

    TGF-β-mediated regulatory T cell induction is not altered in the absence of Drak2 . A) CD4 + CD25 - CD44 lo naïve cells were purified from wildtype and Drak2-/- mice and stimulated with 1μg/ml anti-CD3 and 1μg/ml anti-CD28 with 20ng/ml IL-2 alone or plus 10-fold TGF-β titrations for 3 days. The A) percent and B) number of Foxp3 + cells of electronically gated CD4 + cells is shown. There was no significant difference in the response of the wildtype and Drak2-/- cells according to the Mann-Whitney U -test.

    Journal: PLoS ONE

    Article Title: Drak2 Does Not Regulate TGF-β Signaling in T Cells

    doi: 10.1371/journal.pone.0123650

    Figure Lengend Snippet: TGF-β-mediated regulatory T cell induction is not altered in the absence of Drak2 . A) CD4 + CD25 - CD44 lo naïve cells were purified from wildtype and Drak2-/- mice and stimulated with 1μg/ml anti-CD3 and 1μg/ml anti-CD28 with 20ng/ml IL-2 alone or plus 10-fold TGF-β titrations for 3 days. The A) percent and B) number of Foxp3 + cells of electronically gated CD4 + cells is shown. There was no significant difference in the response of the wildtype and Drak2-/- cells according to the Mann-Whitney U -test.

    Article Snippet: For analysis of phosphorylated Smad2/3, cells were stained with antibodies specific for CD4 and CD8, then fixed with 1X BD Phosflow Lyse/Fix Buffer and permeabilized with BD Phosflow Perm Buffer III according to manufacturer’s instructions (BD Biosciences) and stained with anti-pSmad2/3 antibody (BD Biosciences).

    Techniques: Purification, Mouse Assay, MANN-WHITNEY

    Cytokine production by CD4 + cells isolated from tumor tissue. Tumor tissues were collected from mice (N=3–6 per group, two independent experiments). The CD4 + cells were isolated from tumor tissue using anti-CD4 antibody-coated magnetic beads. Cells were cultured for 48 h with Con A. The concentrations of IFN-γ, IL-4 and IL-10 in cell culture supernatants were measured by ELISA. Differences by mean ± SD were estimated. The statistical significance was calculated (for details see Fig. 3 ).

    Journal: International Journal of Oncology

    Article Title: Treatment with cyclophosphamide supported by various dendritic cell-based vaccines induces diversification in CD4+ T cell response against MC38 colon carcinoma

    doi: 10.3892/ijo.2015.3278

    Figure Lengend Snippet: Cytokine production by CD4 + cells isolated from tumor tissue. Tumor tissues were collected from mice (N=3–6 per group, two independent experiments). The CD4 + cells were isolated from tumor tissue using anti-CD4 antibody-coated magnetic beads. Cells were cultured for 48 h with Con A. The concentrations of IFN-γ, IL-4 and IL-10 in cell culture supernatants were measured by ELISA. Differences by mean ± SD were estimated. The statistical significance was calculated (for details see Fig. 3 ).

    Article Snippet: Lymphocyte stimulation and intracellular staining Splenocytes or CD4+ cells isolated from tumor tissue by anti-CD4 antibody-coated magnetic beads (BD IMag™), were cultured in RPMI supplemented with 10% FBS and 0.5 μg/ml concanavalin A (Con A; Sigma-Aldrich).

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

    Activity of CD4 + cells isolated from tumor tissue. On the 45th day of the experiments, tumor tissues were collected from mice (N=3–6 per group, two independent experiments). The CD4 + cells were isolated from tumor tissue using anti-CD4 antibody-coated magnetic beads. Cells were cultured for 48 h with Con A and the next they were fixed and stained with anti-CD4 antibody and anti-T-bet, anti-GATA3 (A), anti-RORγt, or anti-FoxP3 (B) antibody or appropriate isotype control and analyzed by flow cytometry. The percentage of cells expressing transcription factors among CD4 + cells was estimated by mean ± SD. The statistical significance was calculated (for details see Fig. 3 ).

    Journal: International Journal of Oncology

    Article Title: Treatment with cyclophosphamide supported by various dendritic cell-based vaccines induces diversification in CD4+ T cell response against MC38 colon carcinoma

    doi: 10.3892/ijo.2015.3278

    Figure Lengend Snippet: Activity of CD4 + cells isolated from tumor tissue. On the 45th day of the experiments, tumor tissues were collected from mice (N=3–6 per group, two independent experiments). The CD4 + cells were isolated from tumor tissue using anti-CD4 antibody-coated magnetic beads. Cells were cultured for 48 h with Con A and the next they were fixed and stained with anti-CD4 antibody and anti-T-bet, anti-GATA3 (A), anti-RORγt, or anti-FoxP3 (B) antibody or appropriate isotype control and analyzed by flow cytometry. The percentage of cells expressing transcription factors among CD4 + cells was estimated by mean ± SD. The statistical significance was calculated (for details see Fig. 3 ).

    Article Snippet: Lymphocyte stimulation and intracellular staining Splenocytes or CD4+ cells isolated from tumor tissue by anti-CD4 antibody-coated magnetic beads (BD IMag™), were cultured in RPMI supplemented with 10% FBS and 0.5 μg/ml concanavalin A (Con A; Sigma-Aldrich).

    Techniques: Activity Assay, Isolation, Mouse Assay, Magnetic Beads, Cell Culture, Staining, Flow Cytometry, Cytometry, Expressing

    Activity of CD4 + spleen cells. On the 45th day of the experiments, spleens were obtained from CY ± DC-based vaccine-treated mice (N=3–6 per group, two independent experiments). Splenocytes were stimulated for 48 h with Con A (0.5 μg/ml). For the last 6 h, brefeldin A (10 μg/ml) and monensin (2 μM) were added to cell culture. Stimulated spleen cells were fixed, and stained with anti-CD4 antibody and anti-T-bet, anti-GATA3, anti-FoxP3 antibody. As a control, the appropriate isotypes were used. Samples were analyzed by flow cytometry. The percentage of positive cells among CD4 + splenocytes was calculated (mean ± SD). The statistical significance was calculated (for details see Fig. 3 ).

    Journal: International Journal of Oncology

    Article Title: Treatment with cyclophosphamide supported by various dendritic cell-based vaccines induces diversification in CD4+ T cell response against MC38 colon carcinoma

    doi: 10.3892/ijo.2015.3278

    Figure Lengend Snippet: Activity of CD4 + spleen cells. On the 45th day of the experiments, spleens were obtained from CY ± DC-based vaccine-treated mice (N=3–6 per group, two independent experiments). Splenocytes were stimulated for 48 h with Con A (0.5 μg/ml). For the last 6 h, brefeldin A (10 μg/ml) and monensin (2 μM) were added to cell culture. Stimulated spleen cells were fixed, and stained with anti-CD4 antibody and anti-T-bet, anti-GATA3, anti-FoxP3 antibody. As a control, the appropriate isotypes were used. Samples were analyzed by flow cytometry. The percentage of positive cells among CD4 + splenocytes was calculated (mean ± SD). The statistical significance was calculated (for details see Fig. 3 ).

    Article Snippet: Lymphocyte stimulation and intracellular staining Splenocytes or CD4+ cells isolated from tumor tissue by anti-CD4 antibody-coated magnetic beads (BD IMag™), were cultured in RPMI supplemented with 10% FBS and 0.5 μg/ml concanavalin A (Con A; Sigma-Aldrich).

    Techniques: Activity Assay, Mouse Assay, Cell Culture, Staining, Flow Cytometry, Cytometry

    Tumor tissue infiltration by T lymphocytes. Tumor tissues from CY and cell vaccine-treated mice (N=3–6 per group, two independent experiments) were collected on the 31st, 38th and 45th day of the experiment. The percentage of CD4 + (A) or CD8 + (B) cells among leukocytes (CD45 + ) from tumor tissue. The suspensions of cells from tumor tissue were incubated with anti-CD45 and anti-CD4 or anti-CD8 antibodies and analyzed by flow cytometry. The percentage of CD4 + or CD8 + cells among CD45 + cells was calculated (mean ± SD). The statistical significance ( * P

    Journal: International Journal of Oncology

    Article Title: Treatment with cyclophosphamide supported by various dendritic cell-based vaccines induces diversification in CD4+ T cell response against MC38 colon carcinoma

    doi: 10.3892/ijo.2015.3278

    Figure Lengend Snippet: Tumor tissue infiltration by T lymphocytes. Tumor tissues from CY and cell vaccine-treated mice (N=3–6 per group, two independent experiments) were collected on the 31st, 38th and 45th day of the experiment. The percentage of CD4 + (A) or CD8 + (B) cells among leukocytes (CD45 + ) from tumor tissue. The suspensions of cells from tumor tissue were incubated with anti-CD45 and anti-CD4 or anti-CD8 antibodies and analyzed by flow cytometry. The percentage of CD4 + or CD8 + cells among CD45 + cells was calculated (mean ± SD). The statistical significance ( * P

    Article Snippet: Lymphocyte stimulation and intracellular staining Splenocytes or CD4+ cells isolated from tumor tissue by anti-CD4 antibody-coated magnetic beads (BD IMag™), were cultured in RPMI supplemented with 10% FBS and 0.5 μg/ml concanavalin A (Con A; Sigma-Aldrich).

    Techniques: Mouse Assay, Incubation, Flow Cytometry, Cytometry

    Percentage of T lymphocytes in the spleen. Spleen CD4 + (A) or CD8 + (B) cells from mice treated with CY ± DC-vaccines (N=3–6 per group, two independent experiments) were collected on the 31st, 38th and 45th day of the experiments. The cell suspensions were incubated with anti-CD4 or anti-CD8 antibodies and analyzed by flow cytometry. The percentage of CD4 + or CD8 + cells was not statistically significant.

    Journal: International Journal of Oncology

    Article Title: Treatment with cyclophosphamide supported by various dendritic cell-based vaccines induces diversification in CD4+ T cell response against MC38 colon carcinoma

    doi: 10.3892/ijo.2015.3278

    Figure Lengend Snippet: Percentage of T lymphocytes in the spleen. Spleen CD4 + (A) or CD8 + (B) cells from mice treated with CY ± DC-vaccines (N=3–6 per group, two independent experiments) were collected on the 31st, 38th and 45th day of the experiments. The cell suspensions were incubated with anti-CD4 or anti-CD8 antibodies and analyzed by flow cytometry. The percentage of CD4 + or CD8 + cells was not statistically significant.

    Article Snippet: Lymphocyte stimulation and intracellular staining Splenocytes or CD4+ cells isolated from tumor tissue by anti-CD4 antibody-coated magnetic beads (BD IMag™), were cultured in RPMI supplemented with 10% FBS and 0.5 μg/ml concanavalin A (Con A; Sigma-Aldrich).

    Techniques: Mouse Assay, Incubation, Flow Cytometry, Cytometry