Nsc95397

Fresh Li-Heparin-anticoagulated blood samples were kindly provided by the blood bank of the University of Tübingen. The study is approved by the ethics committee of the University of Tübingen (184/2003 V). The blood was centrifuged at 120 g for 20 min at 21 °C and the platelets and leukocytes-containing supernatant was disposed. Erythrocytes were incubated in vitro at a hematocrit of 0.4% in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO 4 , 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES; pH 7.4), 5 glucose, 1 CaCl 2 , at 37°C for 48 hours. Where indicated, erythrocytes were exposed for 48 hours to NSC-95397 (MedChem Express, Princeton, USA), Ca 2+ -free solution without or with addition of Ca 2+ chelator 1,2-Bis(2aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) BAPTA-AM (Thermo Fisher Scientific, Darmstadt, Germany), necroptosis inhibitor necrostatin-1 (Sigma, Schnelldorf, Germany), p38 kinase inhibitor SB 203580 (Tocris bioscience, Bristol, UK), protein kinase C inhibitor staurosporine (Sigma Aldrich, Hamburg, Germany), casein kinase inhibitor D4476 (Sigma Aldrich, Hamburg, Germany), and pan-caspase inhibitor zVAD (Tocris bioscience, Bristol, UK).