Quantiglo

Human astrocytes were cultured as described above. The complete volume of 10 mL supernatant was collected from each plate, centrifuged to remove particulates and immediately frozen at −80 °C.
For quantitative determination of ET-1 protein a commercial available Elisa-Kit was used (QuantiGlo, human ET-1 Immunoassay, R&D, Wiesbaden, Germany).
To increase the detection limit, the supernatants were lyophilized and resolved in 1 mL of distilled water. To compensate the matrix effect appropriate standards concentrations of ET-1 protein and a blank probe were prepared in 10 mL of supplemented DMEM and analyzed like the samples. The test was performed according to the manufacturer’s manual. Results were obtained using a standard curve generated by a cubic-spline curve fit and were expressed as pg ET-1/10⁶ cells.

For routine laboratory assays blood was obtained at admission to the hospital. It was collected in 6 mL tubes, VACUETTE® Z Serum Clot Activator (Greiner Bio-one GmbH, Kremsmuenster, Austria). The serum aliquots were stored at −80 °C. The Beckman Coulter instrument AU 2700, 2007 (Brea, CA, USA) and Architect c8000, Abbott 2013 (Chicago, IL, USA) were used to measure total plasma cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides and CRP. A specific chemiluminescent ELISA (QuantiGlo; R&D Systems, Wiesbaden-Nordenstadt, Germany) was used to measure IL-6 concentrations and with Human Endothelial Lipase Assay Kit (TaKaRa, Takara Bio Europe S.A.S., Saint-Germain-en-Laye, France) were measured pre-heparin EL protein levels, according to the manufacturer’s instructions.