HUVEC Culture and Resveratrol Effects

Human umbilical vein endothelial cells (HUVECs) were obtained from Cell Applications (Cell Applications, San Diego, CA, USA) and cultured as previously described [43 (link)]. HUVECs were sub-cultured at a split ratio of 1:2 and used within three passages before cell viability was checked using trypan blue. Unless not otherwise specified, cells were plated in 96-well black plates (BD Falcon, Franklin Lakes, NJ) and processed for experiments in Medium M199 containing 5% FCS plus the different treatments. RES 10 mM stock solution was prepared in dimethyl sulfoxide (DMSO); final DMSO concentration in the diluted treatment media was less than 0.1%. DMSO (less than 0.1%) was used as a vehicle control. To investigate the involvement of ROS and NADPH oxidase in the RES-induced cellular effects, in selected experiments, cells were pretreated for 1 h with either 100 µM of the broad antioxidant Tempol [50 (link)] or with 10µM of the flavin-oxidase inhibitor Diphenyleneiodonium (DPI) [49 (link),51 (link),52 (link)]. The involvement of PKC in HUVECs response to RES treatment was investigated by using 2.5 µM of the broad PKC inhibitor chelerythrine (CHE) [53 (link),54 (link)].