Journal: The Journal of Veterinary Medical Science

Article Title: CD4 + T Cell Cytokine Gene and Protein Expression in Duodenal Mucosa of Dogs with Inflammatory Bowel Disease

doi: 10.1292/jvms.13-0008

Figure Lengend Snippet: Sequences of oligonucleotide primers used for quantitative real-time PCR

Article Snippet: Protein levels were determined using commercial ELISA kits specific for canine IL-17A (USCN Life Science, Wuhan, China, 100 μl of supernatants/well), IFN-γ and IL-10 (R&D systems, Minneapolis, MN, U.S.A., 50 μl supernatant/well).

Techniques:

Journal: Cell Communication and Signaling : CCS

Article Title: Deficiency of PI3-Kinase catalytic isoforms p110γ and p110δ in mice enhances the IL-17/G-CSF axis and induces neutrophilia

doi: 10.1186/s12964-017-0185-y

Figure Lengend Snippet: Cytokine concentrations in the serum of WT, p110γ −/− , p110δ −/− and p110γ/δ −/− mice

Article Snippet: Commercial ELISA kits for IL-17A were from Biozol (Biozol, Eching, Germany) and for IL-4, IL-5, G-CSF, CXCL1/KC, CXCL2/MIP-2, and IFN-γ from R&D (R&D Systems, Minneapolis, USA).

Techniques:

Journal: Cell Communication and Signaling : CCS

Article Title: Deficiency of PI3-Kinase catalytic isoforms p110γ and p110δ in mice enhances the IL-17/G-CSF axis and induces neutrophilia

doi: 10.1186/s12964-017-0185-y

Figure Lengend Snippet: Elevated IL-17A levels and increased numbers of IL-17-producing T cells in p110γ/δ −/− mice. IL-17A serum concentrations were measured using a Bio-Plex assay, and IL-17A-producing T cell subsets in the spleen were analyzed by flow cytometry. a Serum concentration of IL-17A in WT, p110γ −/− , p110δ −/− , and p110γ/δ −/− mice ( n = 10). Data were statistically analyzed as described in Fig. . b CD3ε + CD4 + (far-left, top) and CD3ε + CD8 + T cells (far-left, bottom) were gated from pre-gated singlet live NK1.1 − splenic leukocytes and subsequently analyzed for IL-17A expression. Shown are representative examples of the analysis of IL-17A expression in CD3ε + CD4 + and CD3ε + CD8 + T cell subsets from WT, p110γ −/− , p110δ −/− , and p110γ/δ −/− mice. c Percentages of IL-17A + subpopulations of CD3ε + CD4 + or CD3ε + CD8 + T cell subsets in WT, p110γ −/− , p110δ −/− , and p110γ/δ −/− mice ( n = 4–10). Data were statistically analyzed as described in Fig.

Article Snippet: Commercial ELISA kits for IL-17A were from Biozol (Biozol, Eching, Germany) and for IL-4, IL-5, G-CSF, CXCL1/KC, CXCL2/MIP-2, and IFN-γ from R&D (R&D Systems, Minneapolis, USA).

Techniques: Plex Assay, Flow Cytometry, Concentration Assay, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: Deficiency of PI3-Kinase catalytic isoforms p110γ and p110δ in mice enhances the IL-17/G-CSF axis and induces neutrophilia

doi: 10.1186/s12964-017-0185-y

Figure Lengend Snippet: Frequencies of cytokine producing T cell subsets are increased in p110γ/δ −/− mice. To determine frequencies of cytokine producing T helper cell (Th cell) subsets, PMA-stimulated splenocytes were measured by flow cytometry. Th cells were gated as singlet live NK1.1 − CD3ε + CD4 + cells and analyzed for intracellular IL-17A, IFN-γ, IL-4, and IL-5 expression. Cytokine secretion by MACs-purified splenic T cells was determined following incubation with indicated concentrations of plate-coated anti-CD3ε or anti-CD3ε/anti-CD28 and IL-17A, IFN-γ, IL-4, and IL-5 concentrations in the supernatants were measured by ELISA. a Cytokine producing Th cell subsets in the spleens of WT, p110γ −/− , p110δ −/− , and p110γ/δ −/− mice. Flow cytometry plots show representative examples of the analysis of IL-17, IFN-γ, IL-4, and IL-5 expression in CD3ε + CD4 + T cells from one WT and one p110γ/δ −/− mouse and graphs display percentages of cytokine producing Th cell subsets. Bars represent means + SD of pooled data from three independent experiments. b Production of IL-17A, IFN-γ, IL-4, and IL-5 by T cells from WT, p110γ −/− , p110δ −/− , and p110γ/δ −/− mice. Bars represent means + SD of pooled data from three independent experiments carried out in duplicates

Article Snippet: Commercial ELISA kits for IL-17A were from Biozol (Biozol, Eching, Germany) and for IL-4, IL-5, G-CSF, CXCL1/KC, CXCL2/MIP-2, and IFN-γ from R&D (R&D Systems, Minneapolis, USA).

Techniques: Flow Cytometry, Expressing, Purification, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Deficiency of PI3-Kinase catalytic isoforms p110γ and p110δ in mice enhances the IL-17/G-CSF axis and induces neutrophilia

doi: 10.1186/s12964-017-0185-y

Figure Lengend Snippet: IL-17A stimulation of lung tissue cells from p110γ/δ −/− mice induces normal Akt phosphorylation and G-CSF and KC production. To measure IL-17-induced Akt phosphorylation cultivated tissue cells from lungs of WT and p110γ/δ −/− mice were stimulated with 50 ng/mL IL-17A. Cells were harvested at the indicated time points upon which whole cell lysates were subjected to immunoblot analysis using anti-phospho-Akt, anti-Akt, and anti-GAPDH antibodies. IL-17A induced cytokine production of lung tissue cells from WT, p110γ −/− , p110δ −/− , and p110γ/δ −/− mice was measured by ELISA. a Phospho-Akt expression in lung tissue cells from WT and p110γ/δ −/− mice at different time points following IL-17A stimulation. Depicted is a statistical evaluation of phospho-Akt levels and representative blots showing the expression of phospho-Akt, total Akt, and GAPDH. Bars present the average fold change + SD of phospho-Akt levels of unstimulated cells. Phospho-Akt and Akt were first normalized to GAPDH to control for protein loading differences and then phospho-Akt was normalized to Akt levels. Bars represent means + SD of pooled mean values from three independent experiments, each measured in triplicates or quadruplicates. b Dose-dependent production of G-CSF and KC by lung tissue cells from WT, p110γ −/− , p110δ −/− , and p110γ/δ −/− mice that were stimulated with increasing concentrations of IL-17A for 24 h. Bars represent means + SD of pooled mean values from three independent experiments measured in triplicates

Article Snippet: Commercial ELISA kits for IL-17A were from Biozol (Biozol, Eching, Germany) and for IL-4, IL-5, G-CSF, CXCL1/KC, CXCL2/MIP-2, and IFN-γ from R&D (R&D Systems, Minneapolis, USA).

Techniques: Phospho-proteomics, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Control