simplechip human ifn γ promoter primers  (Cell Signaling Technology Inc)

Journal: Oncotarget

Article Title: Cytokine-mediated activation of human ex vivo-expanded Vγ9Vδ2 T cells

doi: 10.18632/oncotarget.17498

Figure Lengend Snippet: (A) Ex vivo-expanded human Vγ9Vδ2 T cells were cultured for 16 h with medium alone, IL-12 (10 ng/mL), IL-18 (10 ng/mL), or IL-12 and IL-18 (10 ng/mL each). Cells were harvested and analyzed to detect the cell-surface of CD3 and Vδ2 TCR and intracellular expression of IFN-γ. Data from healthy donors (n = 5) obtained from independent experiments. Bars represent the mean and standard error of the mean (SEM), **** P < 0.0001, one-way ANOVA, followed by Tukey's multiple comparison test. (B) Ex vivo-expanded Vγ9Vδ2 T cells were stimulated with indicated cytokines for 4, 8, 16 and 20 h and the cells were analyzed in the same way as (A) . (C) Freshly isolated human Vγ9Vδ2 T cells were stimulated and analyzed in the same way as (A) . (D) IL-12/IL-18 treated fleshly isolated Vγ9Vδ2 T cells were further analyzed for the differentiation status based on the cell surface expression of CD27 and CD45RA. Data from healthy donors (n = 3) obtained from independent experiments. Bars represent the mean and SEM, * P < 0.05, one-way ANOVA, followed by Tukey's multiple comparison test.

Article Snippet: The primers were purchased from Cell Signaling Technology (SimpleChIP Human IFN-γ promoter primers #13051) and QIAGEN (EpiTect ChIP qPCR Assay GPH1017290 (−4kb)).

Techniques: Ex Vivo, Cell Culture, Expressing, Isolation

Journal: Oncotarget

Article Title: Cytokine-mediated activation of human ex vivo-expanded Vγ9Vδ2 T cells

doi: 10.18632/oncotarget.17498

Figure Lengend Snippet: Ex vivo-expanded Vγ9Vδ2 T cells were transfected with siRNA targeting IκBζ or control siRNA followed by 16-hour treatment of IL-12 and IL-18. Zombie violet positive cells were gated-out as dead cells. (A) Representative contour plots of IκBζ-positive cells gated on CD3 + Vδ2 TCR + cells. Among three different siRNAs targeting IκBζ, one (s34643) silenced cytokine-induced IκBζ efficiently, and which was used for knocking down IκBζ in further experiments. (B) Representative contour plots and percentage of BTLA-positive cells by CD3 + Vδ2 TCR + cells. (C-F) Representative histogram (left) and gMFI (right) of CD25 (C) , granzyme B (D) , ICAM-1 (E) and IFN-γ (F) by CD3 + Vδ2 TCR + cells. The data were obtained from three independent experiments each using Vγ9Vδ2 T cells from different donors. Bars represent the mean and SEM, * P < 0.05, two-tailed paired Student t test.

Article Snippet: The primers were purchased from Cell Signaling Technology (SimpleChIP Human IFN-γ promoter primers #13051) and QIAGEN (EpiTect ChIP qPCR Assay GPH1017290 (−4kb)).

Techniques: Ex Vivo, Transfection, Two Tailed Test

Journal: Oncotarget

Article Title: Cytokine-mediated activation of human ex vivo-expanded Vγ9Vδ2 T cells

doi: 10.18632/oncotarget.17498

Figure Lengend Snippet: (A) Ex vivo-expanded Vγ9Vδ2 T cells were stimulated with IL-12 alone or in combination with IL-18 for different periods. Representative contour plots and percentage of p-STAT4 positive cells. The data for healthy donors (n = 4) was obtained from independent experiments. Bars represent the mean and SEM, ** P < 0.01, *** P < 0.001, two-way ANOVA, followed by Sidak's multiple comparisons test. (B) Ex vivo-expanded Vγ9Vδ2 T cells were activated with IL-12 and IL-18 for 16 h followed by chromatin immunoprecipitation using anti-STAT4 Ab. Recruitment of STAT4 to the promoter (left) and −4 kb (right) of IFN-γ gene regions were analyzed by qPCR. The data were obtained from five independent experiments each using Vγ9Vδ2 T cells from different donors. Bars represent the mean and SEM, * P < 0.05, two-tailed paired Student t test. (C) Ex vivo-expanded Vγ9Vδ2 T cells were stimulated with IL-18 alone or in combination with IL-12 for 16 h. Representative contour plots and percentage of p-p65 positive cells. The data for healthy donors (n = 3) was obtained from independent experiments. Bars represent the mean and SEM, ** P < 0.01, one-way ANOVA, followed by Tukey's multiple comparison test. (D, E) Ex vivo-expanded Vγ9Vδ2 T cells were transfected with siRNA targeting IκBζ or control siRNA followed by 16-hour treatment of IL-12 and IL-18. Representative contour plots and percentage of p-STAT4- (D) and p-p65- (E) positive cells by CD3 + Vδ2 TCR + cells. The data were obtained from three independent experiments each using Vγ9Vδ2 T cells from different donors. Bars represent the mean and SEM, two-tailed paired Student t test.

Article Snippet: The primers were purchased from Cell Signaling Technology (SimpleChIP Human IFN-γ promoter primers #13051) and QIAGEN (EpiTect ChIP qPCR Assay GPH1017290 (−4kb)).

Techniques: Ex Vivo, Chromatin Immunoprecipitation, Two Tailed Test, Transfection