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, obtained from R&D Systems), following the manufacturer's instructions. Sunrise absorbance reader for microplates (Tecan, Salzburg, Austria) was used to determine optical density for each sample. 2.7. Isolation of Neutrophils, Flow-Cytometry, and RT-PCR We isolated neutrophils from healthy donors buffy coat in order to study surface expression of ETA and ETB by flow-cytometry and the transcripts for ETA and ETB by RT-PCR. Highly purified granulocytes (neutrophils > 96.5%) were isolated and prepared under endotoxin-free conditions using lymphoprep Ficoll-Isopaque. Neutrophils were further enriched by positively removing all contaminating cells with mAb against CD3, CD56, CD19, CD36, CD49d, and Gly-A using a custom-made Easy-Sep kit (StemCell Technologies, Vancouver, BC, Canada) to reach more than 99,7% purity. C646 One million neutrophils were suspended in tubes for FACS analysis. Staining for ETA- and ETB was carried out as already described. RNA extraction and RT-PCR were performed as previously described. 2.8. Analysis of Cytokines and MMP-9 in the Supernatants of Neutrophils Stimulated with ET-1 Neutrophils were seeded in microplates and incubated with or without 100?ng/mL Ultrapure E. coli lipopolysaccharide (LPS) (Invivogen, San Diego, CA) and with or without ET-1. Therefore we used 4 different conditions: (a) neutrophils without any stimulus as negative control, (b) neutrophils incubated with ET-1, (c) neutrophils incubated with LPS alone, (d) and neutrophils incubated with both ET-1 and LPS. Cells were cultured in RPMI-1640-GlutaMAX-I, supplemented with 10% fetal calf serum (FCS), 100?U/mL penicillin, and 100?microg/mL streptomycin. Interleukin-8, TNF-��, Etomidate vascular endothelial growth factor (VEGF), IFN-��, IL-17, and matrix metallopeptidase 9 (MMP-9) released in the supernatants of cultured neutrophils were assessed by ELISA at two different time points (3 and 10 hours). Quantikine Human Immunoassay for the selected molecules was purchased from R&D Systems. Sunrise absorbance reader for microplates was used to determine optical density for each sample. 2.9. Statistical LDN-193189 mouse Analysis All the calculations were performed with SPSS 21.0 statistical package (SPSS Inc., Chicago, IL, USA). All the results are expressed as ��MFI mean �� standard deviation. Quantitative data were assessed using Student's t-test. Correlations between ET-A and ET-B cell surface expression and clinical features were assessed with nonparametric test and multivariate analysis. A value of P
