Lipopolysaccharide induced acute septic shock model A total number of 20 C57 B6 mice at the age of 5 months with an initial body weight of 22 g to 30 g selleck Sunitinib was analyzed in this study. Mice were randomly assigned to 2 groups of control mice and 1 group of LPS treated mice co injected with compound HAK 2. Animals were housed at the Experimental Animal Core Facility of the University of Leipzig. The study was approved by the RegierungsprAsidium Leipzig, License TVV 28 07 on November 14, 2007. To induce septic shock and acute inflammation, 14 mice were injected intraperitoneally with 1 Inhibitors,Modulators,Libraries mg LPS kg body weight. Saline injection i. p. was used as control treatment. Compound HAK 2 was co injected i. p. to 8 LPS treated mice at a concentration of 10 mg kg body weight.

Two hours post treatment mice were sacrificed by CO2 inhalation and tissue samples from hip pocampus Inhibitors,Modulators,Libraries and cortex as well as plasma were prepared as described later. Mouse brain samples for qRT PCR were prepared from 6 8 animals per group. After removal of the brain, the tissue samples were flushed shortly with ice cold saline and placed briefly on filter paper. Cortex and hippocampus were prepared, weighted and snap frozen in liquid nitrogen. Samples were stored at 80 C until RNA isolation. Frozen tissue samples were homogenized in RNA lysis buffer using Precellys ceramic beads. At the end of the experiment blood samples were col lected in ice cooled tubes and centrifuged within the next 20 min. Plasma samples were aliquoted into fractions of 50 ul, shock frozen and stored at 80 C until analysis.

Quantitative real time PCR Total RNA of cultured cells and homogenized mouse brain tissue samples was isolated using RNA isolation kit NucleoSpin RNA Inhibitors,Modulators,Libraries II from Macherey and Nagel. RNA quantity was measured by spectrophotometrical Inhibitors,Modulators,Libraries quantifi cation. Total Inhibitors,Modulators,Libraries RNA was transcribed to cDNA using Super script III and oligo dT primer. Quantitative real time PCR was performed with QuantiFast SYBR Green PCR Master mix using Rotorgene 3000 system. Gene expression was normalized to the expression of three reference genes glyceraldehyde 3 phos phate dehydrogenase, glucose 6 phosphate dehydrogenase and hypoxanthine guanine phos phoribosyltransferase. Primers for PREP were designed using the Primer3 Software. Data were analyzed by Rotorgene 3000 Software version 6. 0 by comparative quantitation.

The appropriate size of PCR products was confirmed by gel electrophoresis stained with ethidium bromide. ELISA Measurements of low IL 6 in conditioned medium and mur ine plasma samples were done by means of human or murine specific IL 6 Cytoset kits according to manufacturers protocols. Preparation of whole cell extracts for western blotting and immunoprecipitation Human U343 cells were lysed with cell lysis buffer supplemented with 0. 2 mg ml sodium orthovanadate, protease inhibitor mix complete mini and 1 mM AEBSF for 30 min on ice.