Interleukin-33 Induces Expression of Adhesion Molecules and ...
Interleukin-33 Induces Expression of Adhesion Molecules and ...
Interleukin-33 Induces Expression of Adhesion Molecules and ...
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S3022). After the cells were washed once with PBS <strong>and</strong> were resuspended in fixative solution<br />
(FACS flow solution, distilled water, BD CellFix TM ), flow cytometric analysis was performed<br />
by FACS Diva s<strong>of</strong>tware (Becton Dickinson). For IL-4, IL-5, IL-10 determination supernatants<br />
were collected from HCAEC incubated in the absence or presence <strong>of</strong> 100 ng/ml IL-<strong>33</strong> for 4,<br />
16 <strong>and</strong> 24 h, respectively. Supernatants were processed per manufacturer`s instruction (BD<br />
Cytometric Bead Array, Human Th1/Th2 cytokine kit). In detail, 25µl conditioned<br />
supernatant was added to 25µl mixed beads (each specific for IL-4, IL-5, IL-10) <strong>and</strong> 25µl PE<br />
detection reagent. The mixture was incubated for 3 h at room temperature in the dark. The<br />
beads were washed <strong>and</strong> analyzed immediately by flow cytometry. Mean fluorescence<br />
intensities (MFI) for treated endothelial cells were compared to the MFI <strong>of</strong> unstimulated cells.<br />
Total RNA purification <strong>and</strong> cDNA preparation<br />
Cells were treated as described, supernatants were removed <strong>and</strong> total cellular RNA was<br />
isolated using High Pure RNA Isolation Kit (Roche, Basel, Switzerl<strong>and</strong>) according to the<br />
manufacturer’s instructions. For IL-<strong>33</strong> mRNA <strong>and</strong> ST2 mRNA measurement, three<br />
representative samples (each 25 mg <strong>of</strong> wt) were collected from each carotid artery<br />
lesion.Frozen tissue was homogenized using a ball mill (Retsch, Haan, Germany), <strong>and</strong> mRNA<br />
was isolated usingHigh Pure RNA Tissue Kit (Roche). Reverse transcription was performed<br />
using Transcriptor First Str<strong>and</strong> cDNA Synthesis Kit (Roche).<br />
RealTime Polymerase Chain Reaction<br />
RealTime-PCR was performed using LightCycler® TaqMan® Master (Roche) according to<br />
the manufacturer’s instructions. Primers were designed using the Roche Universal<br />
ProbeLibrary Assay Design Centre (http://www.universalprobelibrary.com/): GAPDH<br />
(forward primer: 5’-agccacatcgctcagacac-3’, reverse primer: 5’-gcccaatacgaccaaatcc-3’,<br />
UPLprobe #60; Amplicon Size [bp] 66) – ST2 (forward primer: 5’-ttgtcctaccattgacctctacaa-3’,<br />
Downloaded from<br />
http://atvb.ahajournals.org/ at Bibliothek der MedUniWien (IX0000096057) on September 2, 2011
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