Human umbilical vein ecs huvecs

Cells were cultured at 37°C in 5% CO₂. Human dermal microvascular ECs (HDMVECs) and human umbilical vein ECs (HUVECs) were obtained from Lonza (Allendale, NJ). Primary human brain microvascular ECs (HBMECs) were obtained from ScienCell (Carlsbad, CA). A human brain microvascular cell line, CMEC/D3 [Daniels et al., 2013 (link)], was tested, but the cells did not express VE-cadherin and did not form monolayers, so they were not included in this study. Primary ECs were cultured in EBM-2 base medium supplemented with the EGM-2 kit for HUVECs or the EGM-2 MV kit for HDMVECs and HBMECs. HBMECs were not used after four passages; HDMVECs and HUVECs were not used after nine passages. Human PBLs were isolated as described [Mooren et al., 2014 (link)]. Human NK-92 cells (ATCC, Manassas, VA) were cultured as described [Mukherjee et al., 2014 ]. Human 92.1 uveal melanoma cells, a generous gift of Dr. Martine Jager (Laboratory of Ophthalmology, Leiden University), were grown in RPMI 1640 medium (Life Technologies, Carlsbad, CA) supplemented with 10% FBS and antibiotics.

Human Umbilical Vein ECs (HUVECs) and Human Micro Vascular ECs (HMVECs) (both from Lonza) were grown in EGM-2 (EBM-2 medium supplemented with growth factors and normal 5mM D-Glucose, NG) and 2% Foetal Bovine Serum (FBS) (Lonza). To mimic diabetes and ischemia in vitro, the ECs were maintained in EBM-2 (growth factor free medium) with 2% FBS and 25 mM D-glucose (high glucose, HG). L-Glucose was used as an osmotic control (Cont) at the concentration of 25 mM. HUVECs were used between P2 and P6 passage. HA-1077 or Y27632 (Sigma) have been used at concentration of 10μM; Eptifibatide (Sigma) has been used at concentration of 250 μM. HEK293T cells (ATCC®, CRL-11268) were cultured in D-MEM with 10% FBS (Life Technologies). Pericytes were grown on fibronectin (10 μg/mL) coated plates and maintained in EGM-2 with 2% FBS.