Journal: Clinical and Developmental Immunology

Article Title: Cysticerci Drive Dendritic Cells to Promote In Vitro and In Vivo Tregs Differentiation

doi: 10.1155/2013/981468

Figure Lengend Snippet: In vitro differentiation of dendritic cells in the presence of Taenia solium or T. crassiceps cysticerci promotes Treg induction. (a) Increased percentage in Treg cell induction. Percentage was calculated as follows: (percentage of Tregs induced/percentage of basal Tregs) × 100. Different letters indicate significant differences between groups at P < 0.05. (b) Representative histograms showing Foxp3 induction within CD4 + CD25 high cells. Data are representative of three independent experiments.

Article Snippet: A RosetteSep Human CD4 + T Cell Enrichment Cocktail kit was used for CD4 + T cell purification (StemCell, Vancouver, Canada).

Techniques: In Vitro

Journal: Clinical and Developmental Immunology

Article Title: Cysticerci Drive Dendritic Cells to Promote In Vitro and In Vivo Tregs Differentiation

doi: 10.1155/2013/981468

Figure Lengend Snippet: Levels of cytokines in supernatants from in vitro DC differentiation and in vitro regulatory T cell induction.

Article Snippet: A RosetteSep Human CD4 + T Cell Enrichment Cocktail kit was used for CD4 + T cell purification (StemCell, Vancouver, Canada).

Techniques: In Vitro

Journal: Clinical and Developmental Immunology

Article Title: Cysticerci Drive Dendritic Cells to Promote In Vitro and In Vivo Tregs Differentiation

doi: 10.1155/2013/981468

Figure Lengend Snippet: Phenotype of peripheral dendritic and regulatory T cells in NC patients and healthy subjects.

Article Snippet: A RosetteSep Human CD4 + T Cell Enrichment Cocktail kit was used for CD4 + T cell purification (StemCell, Vancouver, Canada).

Techniques:

Journal: Cells

Article Title: Glycogen Synthase Kinase-3β Facilitates Cytokine Production in 12-O-Tetradecanoylphorbol-13-Acetate/Ionomycin-Activated Human CD4 + T Lymphocytes

doi: 10.3390/cells9061424

Figure Lengend Snippet: A combination of 12-O-tetradecanoylphorbol-13-acetate (TPA) and ionomycin causes in vitro activation of CD4 + T lymphocytes. ( A ) Representative dot plots of immunostaining followed by flow cytometric analysis detected the IFN-γ expression in CD4 + and CD8 + T lymphocytes 6 h posttreatment with TPA plus ionomycin (T/I). In addition, ELISA determined the levels of IFN-γ in the cell supernatants of TPA-, ionomycin (Iono)-, and T/I-treated CD4 + and CD8 + T lymphocytes ( B ) and Jurkat T cells ( C ). ( D ) Representative dot plots and histograms of immunostaining followed by flow cytometric analysis detected the IFN-γ expression in T/I-stimulated murine CD4 + CD8 − (a), CD4 + CD8 + (b), and CD4 − CD8 + (c) thymocytes. Primary CD4 + T cells were obtained from three individual volunteers of number (No.) 1 to 3. ELISA determined the levels of cytokines in the cell supernatants of TPA-, ionomycin-, or T/I-treated CD4 + T cells 6 h posttreatment ( E ) without or ( F ) with Bis (5 μM) and BAPTA (10 μM) pretreatment for 0.5 h. An LDH assay was used to detect cell cytotoxicity, and the results are normalized to the untreated group. For flow cytometric analysis, the percentages of IFN-γ-expressing cells are shown. For ELISA, the data are shown as the mean ± SD from three individual experiments. * p < 0.05 and *** p < 0.001 compared to untreated cells. # p < 0.05, ## p < 0.01, and ### p < 0.001 compared to the T/I-treated group. ns, not significant.

Article Snippet: Following PBMC isolation, human CD4 + and CD8 + T cells were individually separated using the BD IMagTM Human CD4 or CD8 Lymphocyte Enrichment Set-DM kits according to the manufacturer’s instructions ( https://www.bdbiosciences.com/us/reagents/research/magnetic-cell-separation/human-cell-separation-reagents/human-cd4-t-lymphocyte-enrichment-set-dm/p/557939 ).

Techniques: In Vitro, Activation Assay, Immunostaining, Expressing, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay

Journal: Cells

Article Title: Glycogen Synthase Kinase-3β Facilitates Cytokine Production in 12-O-Tetradecanoylphorbol-13-Acetate/Ionomycin-Activated Human CD4 + T Lymphocytes

doi: 10.3390/cells9061424

Figure Lengend Snippet: Treatment with T/I activates GSK-3β and then induces GSK-3β-regulated cytokine production. ( A ) Western blotting detected the phosphorylation of GSK-3β (47 kDa) in CD4 + lymphocytes treated with the T/I combination. ( B ) Primary CD4 + T cells were obtained from three individual volunteers of number (No.) 1 to 3. ELISA determined the levels of cytokines in the cell supernatants of T/I-treated CD4 + T lymphocytes and Jurkat T cells 6 h posttreatment after BIO (10 μM) pretreatment for 0.5 h. DMSO was used as a control. ( C ) Western blot analysis showed GSK-3β (47 kDa) knockdown in Jurkat T cells (shGSK-3β). Luciferase shRNA (shLuc) was used as a control. ( D ) ELISA determined the levels of cytokines in the shGSK-3β-transfected cells pretreated with BIO (10 μM) for 0.5 h and then stimulated with or without T/I for an additional 6 h. For Western blotting, β-actin (42 kDa) was used as an internal control. The ratio of phosphorylated protein/total protein/β-actin is shown. For ELISA, an LDH assay was used to detect cell cytotoxicity, and the results are normalized to the untreated group. The data are shown as the mean ± SD from three individual experiments. *** p < 0.001 compared to the untreated cells. # p < 0.05, ## p < 0.01, and ### p < 0.001 compared to the T/I-treated group. ns, not significant.

Article Snippet: Following PBMC isolation, human CD4 + and CD8 + T cells were individually separated using the BD IMagTM Human CD4 or CD8 Lymphocyte Enrichment Set-DM kits according to the manufacturer’s instructions ( https://www.bdbiosciences.com/us/reagents/research/magnetic-cell-separation/human-cell-separation-reagents/human-cd4-t-lymphocyte-enrichment-set-dm/p/557939 ).

Techniques: Western Blot, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Control, Knockdown, Luciferase, shRNA, Transfection, Lactate Dehydrogenase Assay

Journal: Cells

Article Title: Glycogen Synthase Kinase-3β Facilitates Cytokine Production in 12-O-Tetradecanoylphorbol-13-Acetate/Ionomycin-Activated Human CD4 + T Lymphocytes

doi: 10.3390/cells9061424

Figure Lengend Snippet: Treatment with T/I induces Pyk-2-regulated GSK-3β activation and cytokine production. ( A ) Western blotting detected the phosphorylation of GSK-3β (47 kDa) in CD4 + lymphocytes treated with the T/I combination in the presence of A9 (10 μM). ( B ) ELISA determined the levels of cytokines in the cell supernatants of T/I-treated CD4 + T lymphocytes and Jurkat T cells 6 h posttreatment after A9 pretreatment for 0.5 h. For Western blotting, β-actin (42 kDa) was used as an internal control. The ratio of phosphorylated protein/total protein/β-actin is shown. For ELISA, an LDH assay was used to detect cell cytotoxicity, and the results are normalized to the untreated group. The data are shown as the mean ± SD from three individual experiments. *** p < 0.001 compared to the untreated cells. ## p < 0.01 and ### p < 0.001 compared to the T/I-treated group. ns, not significant.

Article Snippet: Following PBMC isolation, human CD4 + and CD8 + T cells were individually separated using the BD IMagTM Human CD4 or CD8 Lymphocyte Enrichment Set-DM kits according to the manufacturer’s instructions ( https://www.bdbiosciences.com/us/reagents/research/magnetic-cell-separation/human-cell-separation-reagents/human-cd4-t-lymphocyte-enrichment-set-dm/p/557939 ).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Control, Lactate Dehydrogenase Assay

Journal: Cells

Article Title: Glycogen Synthase Kinase-3β Facilitates Cytokine Production in 12-O-Tetradecanoylphorbol-13-Acetate/Ionomycin-Activated Human CD4 + T Lymphocytes

doi: 10.3390/cells9061424

Figure Lengend Snippet: Treatment with T/I induces GSK-3β activation and cytokine production through calcineurin (PP2B) signaling. ( A ) Western blotting detected the phosphorylation of GSK-3β (47 kDa) in CD4 + lymphocytes treated with the T/I combination in the presence of cyclosporine A (CsA, 10 μM). CD4 + lymphocytes were pretreated with Bis, BAPTA, CsA, VIVIT, CAPE, PD98059, or a combination of VIVIT, CAPE, and PD98059 for 0.5 h and then stimulated with T/I for 6 h. ( B ) The PP2B activity assay detected the phosphatase activity. ( C , D , and E ) ELISA determined the levels of cytokines in the cell supernatants of the T/I-treated cells. For Western blotting, β-actin (42 kDa) was used as an internal control. The ratio of phosphorylated protein/total protein/β-actin is shown. For ELISA, an LDH assay was used to detect cell cytotoxicity, and the results are normalized to the untreated group. The data are shown as the mean ± SD from three individual experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the untreated cells. ### p < 0.001 compared to the T/I-treated group. ns, not significant.

Article Snippet: Following PBMC isolation, human CD4 + and CD8 + T cells were individually separated using the BD IMagTM Human CD4 or CD8 Lymphocyte Enrichment Set-DM kits according to the manufacturer’s instructions ( https://www.bdbiosciences.com/us/reagents/research/magnetic-cell-separation/human-cell-separation-reagents/human-cd4-t-lymphocyte-enrichment-set-dm/p/557939 ).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Activity Assay, Enzyme-linked Immunosorbent Assay, Control, Lactate Dehydrogenase Assay

Journal: Cells

Article Title: Glycogen Synthase Kinase-3β Facilitates Cytokine Production in 12-O-Tetradecanoylphorbol-13-Acetate/Ionomycin-Activated Human CD4 + T Lymphocytes

doi: 10.3390/cells9061424

Figure Lengend Snippet: Treatment with a GSK-3β inhibitor reduces T/I-induced mortality and cytokine production in vivo. ( A ) Survival rate of mice following T/I treatment with or without BIO therapy. Six groups were included: PBS (n = 3 for control), T200 (n = 3; TPA, 200 μg/kg), T50/I (n = 3; TPA, 50 μg/kg plus ionomycin, 250 μg/kg), T100/I (n = 5; TPA, 100 μg/kg plus ionomycin, 250 μg/kg), T200/I (n = 6; TPA, 200 μg/kg plus ionomycin, 250 μg/kg), and T200/I/BIO (n = 12; TPA, 200 μg/kg plus ionomycin, 250 μg/kg plus BIO 2 mg/kg). ( B and C ) Mice were pretreated with BIO (2 mg/kg) for 0.5 h and were then stimulated with T/I (n = 3; TPA, 200 μg/kg plus ionomycin, 250 μg/kg) for the indicated time. ELISA determined the levels of IFN-γ and TNF-α in the mouse serum. The data are shown as the mean ± SD obtained from three mice. * p < 0.05 and *** p < 0.001 compared with the PBS-treated group. ## p < 0.01 and ### p < 0.001 compared with the T/I-treated group. ( D ) A model of GSK-3β-regulated cytokine production in T/I-activated human CD4 + T lymphocytes.

Article Snippet: Following PBMC isolation, human CD4 + and CD8 + T cells were individually separated using the BD IMagTM Human CD4 or CD8 Lymphocyte Enrichment Set-DM kits according to the manufacturer’s instructions ( https://www.bdbiosciences.com/us/reagents/research/magnetic-cell-separation/human-cell-separation-reagents/human-cd4-t-lymphocyte-enrichment-set-dm/p/557939 ).

Techniques: In Vivo, Control, Enzyme-linked Immunosorbent Assay