Egm 2 singlequot kit suppl growth factors

HUVEC were purchased from ATCC (Manassan, VA). HUVEC were maintained in EBM-2 basal medium (Lonza, Basel, Switzerland) supplemented with EGM-2 Single Quot Kit Suppl. & Growth Factors (Lonza). HUVECs (2.5 × 10⁴ cells) were cultured in OptiPlate™-96F microplate (Perkin Elmer, Waltham, MA). Cells were incubated in the medium with or without BTX-A (0, 0.1, 0.5, 1.0 U/ml FBS(-) DMEM) at 37°C for 24 hours. Cells were stimulated with 0.25 mM H₂O₂ (100 μl/well) for 2 hours, and then ROS levels were measured with DCFDA Cellular ROS Detection Assay Kit (abcam) according to the manufacturer's protocol. Fluorescence was detected by plate reader (Perkin Elmer).

The human retinoblastoma cell lines (Weri-Rb1 and Y79), ARPE-19 (human retinal pigment epithelial cell line) and human retinal microvascular endothelial cells (HRMECs) were purchased from the Shanghai Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences. Weri-Rb1 and Y79 were cultured with RPMI 1640 medium (Gibco, U.S.A.) and supplemented with 10% fetal bovine serum (FBS, Gibco, U.S.A.). ARPE-19 was maintained in DMEM medium (Gibco, U.S.A.) with 10% FBS (Gibco, U.S.A.). HRMECs were maintained in EGM-2 SingleQuot Kit Suppl. & Growth Factors (Lonza, U.S.A.). All cell lines were incubated in a humidified chamber at 37°C with 5% CO₂.

Primary HUVECs (human umbilical vein endothelial cells) were isolated from human umbilical cord provided by Department of Gynaecology and Obstetrics of the University Hospital Aachen in accordance with the human subjects approval of the local ethics committee of the RWTH Aachen University Hospital (votum #EK 2067) after obtaining written consent. The approval includes the use of umbilical cord-derived cells for in vitro studies in tissue engineering. The umbilical cord was washed with 37°C Phosphate Buffered Saline (PBS) and was cannulated. 2.4 IU/mL Dispase-solution (Roche^®) was filled into the vein. The vein was incubated for 30 min at 37°C and 5% CO₂. Detached HUVECs were flushed with 37°C pre-warmed Phosphate Buffer Saline (PBS) into a 50 mL falcon tube (Falcon BD) and centrifuged at 500 g for 5 min. The supernatant was discarded and the cell pellet was resuspended in EBM-2 (Lonza^®) supplemented with EGM-2 SingleQuot Kit Suppl. & Growth Factors (Lonza^®) and 1% antibiotic/antimycotic solution (Gibco^®). The resuspended cells were plated into cell culture flasks (75 cm³, Greiner bio-one), pre-coated with gelatine (Gelatine Type B, Sigma) and were maintained in a humidified incubator at 37°C and 5% CO₂ and cultivated to 80% confluency. For all experiments, pooled cells from four different donors were used in passage four.