Adenosine 5 diphosphate sodium salt adp

Blood was collected from healthy donor volunteers who provided informed consent (HaemoScan BV, Groningen, The Netherlands) and buffered with sodium citrate. To assess platelet aggregation (impedance method), blood was diluted with 0.9% NaCl continuously mixed with a stir bar, impedance electrodes were fixed into the blood-containing tubes, and indicated mAbs, adenosine 5′-diphosphate sodium salt (ADP; Sigma-Aldrich) or vehicle (10 mM L-Histidine pH 5.5 containing 0.1 M sodium chloride) were added to the blood suspension. Aggregation was measured for 6 min. The maximum slope of the aggregation curve for the first 3 min was determined from the recordings by “R: A language and environment for statistical computing” (R Foundation for Statistical Computing). To assess platelet activation, the blood suspension was incubated with indicated mAbs, arachidonic acid (Sigma-Aldrich) or vehicle for 1 h at 37 °C. Samples were centrifuged and plasma was collected to execute the thromboxane B2 enzyme immunoassay (Cayman Chemical).

3-carbamoyl-2,2,5,5-tetramethyl-1-pyrrolidine-1-oxyl (carbamoyl-PROXYL) was purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). Rotenone was purchased from MP Biomedicals (Santa Ana, CA, USA). Succinate and KCN were purchased from Wako Pure Chemical Industries (Osaka, Japan). Adenosine 5′-diphosphate sodium salt (ADP) was purchased from Sigma-Aldrich (St Louis, MO, USA). 8-OHdG was purchased from Bioss Antibodies (Woburn, MA, USA). 4-hydroxy-2-neonal monoclonal antibody was purchased from JaICA (Shiduoka, Japan). All other chemicals were commercially available, and of reagent grade quality.

Anti-TOM20 (#sc-11415, 1:200 for immunofluorescence staining) and anti-NQO1 (#sc-32793, 1:1000 for immunoblotting) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#10494-1-AP, 1:20,000 for immunoblotting) was purchased from Proteintech (Rosemont, IL, USA). Alexa Fluor 488 (#A11001) or Alexa Fluor 700 (#A21036)–conjugated secondary antibodies, Alexa Fluor 488–conjugated phalloidin (#A12379), and 4′,6-diamidino-2-phenylindole (DAPI) (#1306) were obtained from Invitrogen (Carlsbad, CA, USA). IRDye800CW-labeled anti-rabbit secondary antibody (#926-32211) was purchased from LI-COR Biosciences (Lincoln, NE, USA). Isoplumbagin (5-hydroxy-3-methyl-1,4-naphthoquinone) was acquired from AKos GmbH (Steinen, Germany). Oligomycin (#75351), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (#C2920), rotenone (#R8875), antimycin A (#A8674), lactobionic acid (#153516), taurine (#T8691), digitonin (#D141), glutamate (#G8415), malate (#M1000), succinate (#S3674), adenosine 5′-diphosphate sodium salt (ADP) (#A2754), TMPD (#T3134), and ascorbate (#A4034) were purchased from Sigma-Aldrich (St Louis, MO, USA).