biotinylated polyclonal goat antihuman il 1α ab  (R&D Systems)

Journal: Cells

Article Title: Fisetin, a 3,7,3′,4′-Tetrahydroxyflavone Inhibits the PI3K/Akt/mTOR and MAPK Pathways and Ameliorates Psoriasis Pathology in 2D and 3D Organotypic Human Inflammatory Skin Models

doi: 10.3390/cells8091089

Figure Lengend Snippet: Fisetin suppresses the expression and secretion of IFN-γ and IL-17 in activated human peripheral blood mononuclear cells (PBMC). PBMC were prepared from the circulating blood from three different healthy blood donors, and were cultured without (resting) or with anti-CD3 plus anti-CD8 (a-CD3/a-CD28), either treated with 10 µM of fisetin or the vehicle alone (-) for 10 min before activation. ( A ) PBMC were activated for 6 h before measurement of the type-1 and type-17 proinflammatory cytokines IFN-γ ( IFNG ) and IL-17A ( IL17A ) mRNA expression levels. Fold change in mRNA levels compared to resting cells, whose expression level was fixed at 1, were determined. Means ± SE are shown, and log10 transformation followed by paired t -test was used to compare mRNA levels in a-CD3/a-CD28-activated PBMC with or without fisetin. ( B ) PBMC were activated for 48 h in the indicated conditions and secreted cytokines (IFN-γ, IL-17A, and IL-4) present in the cell culture media were measured by ELISA. Means ± SD are shown and the paired t -test was used to compare values between fisetin and no fisetin (–) for a-CD3/a-CD28-activated PBMC.

Article Snippet: Recombinant human (rh) IL-22, IL-17A, TNF-α, anti-CD3, anti-CD28, and biotinylated polyclonal goat antihuman IL-17A were from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Cell Culture, Activation Assay, Transformation Assay, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Fisetin, a 3,7,3′,4′-Tetrahydroxyflavone Inhibits the PI3K/Akt/mTOR and MAPK Pathways and Ameliorates Psoriasis Pathology in 2D and 3D Organotypic Human Inflammatory Skin Models

doi: 10.3390/cells8091089

Figure Lengend Snippet: Fisetin modulated the expression of makers of psoriasis-like inflammation and mTOR activity in 3D human skin model of psoriasis. Immunofluorescence staining for p-p70S6K and Psoriasin was performed on FTRHSP sections by incubating with primary antibody against targets overnight at 4 °C followed by incubation with specific Alexa Flour 488-labeled secondary antibodies for 2 h at room temperature in the dark. Samples were mounted in mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) and analyzed microscopically. ( A – E ) the antimicrobial peptide (psoriasin) and mTOR activation effector, p-p70S6K stainings are shown in red and green, respectively, and DAPI in blue. Representative pictures are shown. Scale bar = 20 μm. The yellow arrows delineate the thickness of the stratum corneum, while the white arrows indicate the thickness of the viable epidermis. ( F , G ) For densitometric quantification, each color image was separated into its green, blue, and red channel components using ImageJ software (National Institutes of Health, Bethesda, MD, USA), and green and red channels were used to analyze mean fluorescent intensity. Data shown here are mean fluorescence intensity ± SEM. ( H ) Differential protein expression levels of IL-17A secreted in the conditioned media of FTRHSP cultures. Pro-inflammatory cytokines IL-17A in conditioned media, were analyzed when 3D FTRHSP were treated without and with fisetin (10–30 µM) treatment or 0.1 µM Vit-D 3 for five days following activation by co-culturing with anti-CD3/CD28 activated CD4+ T cells for seven days as described in . Significant differences were determined and significance of comparisons were made for the expression levels of the target proteins of FTRHSP tissues versus fisetin or Vitamin D 3 -treated FTRHSP, corresponding to the solid lines, and also between nonactivated T cell-exposed control RHSE versus FTRHSP tissues, indicated by the broken lines, as denoted by * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Recombinant human (rh) IL-22, IL-17A, TNF-α, anti-CD3, anti-CD28, and biotinylated polyclonal goat antihuman IL-17A were from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Activity Assay, Immunofluorescence, Staining, Incubation, Labeling, Activation Assay, Software, Fluorescence