Prior to use, S1P was dissolved in 4 mg/mL fatty acid-free BSA solution. Protease inhibitors Complete and Pefabloc SC were obtained from Roche Applied Science (Mannheim, Germany), while phosphatase inhibitor okadaic selleck chemicals acid was from Alexis Biochemicals (Grünberg, Germany). Mononuclear cells were routinely isolated from citrated blood of healthy single donor by pancoll (PanBiotech, Aidenbach, Germany) density centrifugation

and counter flow elutriation as described previously 40. The resulting monocyte fraction consisted of more than 97% pure monocytes. Cells were cultured in RPMI 1640 supplemented with 5% FBS and 2 mM L-glutamine (all from Biochrom (Berlin, Germany)) and treated with stimuli and inhibitors essential as published earlier 3. Approval for these studies was obtained from the Institutional Review board at the University of Lübeck (Lübeck, Germany), and informed consent was provided according to the Declaration of Helsinki. Generation of ROS was determined in a microplate luminometer (LB 96V; Berthold (Wildbach, Germany)) by measurement of chemiluminescence in the presence of 60 μg/mL luminol Ixazomib nmr (5-amino-2,3-dihydro-1,4-phthalazindione; Roche Applied Science) essentially as described

elsewhere 2, 3. In brief, monocytes were stimulated with 4 μM CXCL4 or increasing concentrations of S1P and chemiluminescence was recorded for 60 min. Individual assay backgrounds were determined in samples of unstimulated cells

in the presence or absence of inhibitors run in parallel and were substracted. Data were expressed as relative light units and quantified by integration over the time periods indicated. Determination of apoptotic and necrotic cells was done by double labeling with annexin V-FITC and PI, according to manufacturer’s recommendations (Bender MedSystems, Heidelberg, Germany) 3. The populations of apoptotic and necrotic cells were defined by their characteristic binding patterns annexin Vhigh/PIlow, and annexin Vhigh/PIhigh, respectively. Phosphorylated Erk MAPK was detected in cell lysates by western blot analysis with antibodies specific for the phosphorylated (activated) kinases essential PLEK2 as described earlier 3. Proteins derived from cell lysates (40 or 80 μg/lane) were separated by SDS-PAGE 41 using 12% polyacrylamide gels and blotted onto polyvinylidene fluoride membranes (Roth). Immunodetection was performed as described in detail elsewhere 3, 42. Band intensities on blot membranes were quantified using Odyssey software 2.1 and presented either as integrated intensities or fold induction from unstimulated control. Total RNA was purified using NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany) according to manufacturer’s recommendations followed by reverse transcription into cDNA using First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany).