Our small-scale display identified the anti-epilepsy drug Phenhydan? as a potent inhibitor of death receptor-induced necroptosis and apoptosis. for inflammation-driven diseases caused by aberrant cell death. with the approval of German and French local authorities. Ischemia-reperfusion injury Induction of murine kidney IRI was performed via a midline abdominal incision and bilateral renal pedicle clamping for 47?min using microaneurysm clamps (Aesculap, Inc., Center Valley, PA USA). Throughout the surgical procedure, the body heat of the mice was managed at between 36?C and 37?C by continuous monitoring using a temperature-controlled self-regulated heating system Rhoa (Fine Science Tools GmbH, Heidelberg, Germany). After removal of the clamps, reperfusion of the kidneys was confirmed visually. The stomach was closed in two layers using standard 6-0 sutures. To maintain fluid balance, all of the mice were supplemented with 1?ml of prewarmed PBS administered intraperitoneally directly after surgery. The mice were sacrificed 48?h after reperfusion. Serum creatinine and urea values were measured in the Central laboratory of the University or college Hospital Schleswig-Holstein, Kiel (Germany). IRI experiments were performed in a double-blinded manner. Where indicated, Phenhydan? was added at a final concentration of 1 1?mM to the drinking water (renewed every day) of the mice 7 days before the onset of ischemia and until the end of the reperfusion phase. In vivo model of liver injury C57BL/6 mice were subjected to ConA or saline treatment and sacrificed 7?h later. ConA was administered intravenously at a concentration of 15?mg/kg body weight. Where indicated, a singular injection of Phenhydan? was administered intraperitoneally at a concentration of 34?mg/kg body weight (total volume per mouse was 200?l) 30?min before ConA administration. Appropriate amount of PBS (200?l per mouse) was applied as vehicle control. The plasma concentrations of alanine aminotransferase, aspartate aminotransferase, and ABT-639 lactate dehydrogenase were measured in the Central laboratory of the Universit Pierre et Marie Curie, Paris (France) as well as in the Central laboratory of the University or college Hospital Schleswig-Holstein, Kiel (Germany). Histologic, immunohistochemical, and morphologic assessments Kidney and liver biopsies were fixed in 4% neutral-buffered formaldehyde and embedded in paraffin. The 3-m sections produced were dewaxed, rehydrated, and subjected to periodic acid-Schiff staining (kidney) and haematoxylin and eosin (liver) according to routine protocols. Stains were evaluated blinded by an experienced pathologist. Sections were evaluated using an U-DO3 microscope (Olympus Corp., Tokyo, Japan). Representative photomicrographs were taken using a Zeiss system (an Axioplan microscope with an MRT digital camera and Axiovision Software; Carl Zeiss AG, Oberkochen, ABT-639 Germany). To analyze cell death of the tissue sections, the TdT-mediated dUTP nick end labeling (TUNEL) assay was performed using the fluorescence detection kit according to the manufacturers instructions (Promega, Mannheim, Germany). Briefly, tissue sections were dewaxed, rehydrated, fixed in ABT-639 4% paraformaldehyde and permeabilized with Proteinase K for 10?min at RT. Following this, the sections were equilibrated with the provided buffer for 10?min and labeled with the TdT reaction mix for 60?min at 37?C in a humidified dark environment. To stop the labeling reactions, sections were incubated with the provided stopping buffer for 15?min at RT in the dark. The sections were then washed with PBS for 5?min. Finally, the sections were mounted with fluorescence-mounting media (Dako, Glostrup, Denmark) made up of 4,6-Diamidin-2-phenylindol (DAPI) for cell nuclei counterstaining. Fluorescence micrographs were taken using Zeiss Axio Imager Z1 fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany) at magnifications of 20 and 40 magnification using a standard fluorescein filter set to view the green fluorescence at 520?nm, and blue fluorescence of DAPI at 380?nm. Quantification of TUNEL-positive cells was performed manually by two blinded observers and reproduced in triplicate by each of them. Complex-I purification MEFs were seeded in 15-cm dishes and treated as indicated with 3x FLAG-TNF (5?mg/ml). The medium was removed and the plates were washed with ice-cold PBS. Then, the plates were frozen at?80?C. The plates were later thawed on ice and the cells were lysed in 1% Triton X-100 lysis buffer (30?mM Tris-HCl [pH 7.4], 120?mM NaCl, 2?mM EDTA, 2?mM KCl, 10% glycerol, and 1% Triton X-100).