Ebm 2 plus egm 2 mv supplements

Fresh human cord blood, donated under full ethical approval by healthy volunteers at the Northern Ireland Blood Transfusion Service (Belfast, U.K.), underwent density gradient fractionation for the isolation of mononuclear cells and was selected for ECFCs via resuspension in complete medium (EBM-2 plus EGM-2 MV supplements, Lonza Group) supplemented with 10% FBS and seeding onto 24-well culture plates precoated with rat tail collagen type 1 (BD Biosciences, Bedford, U.K.) at a density of 1 × 10⁷ cells/mL. Cells were labeled (Qtracker 655, Invitrogen, Life Technologies, Carlsbad, CA) per the manufacturer’s instructions.

In vitro measurements of transendothelial electrical resistance (TER) were performed with an electrical cell substrate impedance sensing (ECIS) system (Applied Biophysics, Troy, NY). Human retinal microvascular endothelial cells (HrMVECs) (Cell Systems, Kirkland, WA) were seeded (50,000 cells/well) onto fibronectin-coated gold microelectrodes in ECIS culture wells (8W10E+; Applied Biophysics) and incubated overnight at 37°C in complete medium (EBM-2 plus EGM2-MV supplements, Lonza Group, Basel, Switzerland) until cell resistance reached a plateau. Cells were serum starved for 1 h until resistance was stabilized (1,200 Ω). Each well received one of three experimental treatments: COMP-Ang1 protein (Enzo Life Sciences, Farmingdale, NY), VEGF protein (R&D, Minneapolis, MN), or PBS. Monitoring was continued for 21 h. The data from triplicate wells were averaged and presented as normalized resistance versus time.