C 22211

Human pulmonary arterial endothelial cells (PAECs) were purchased from PromoCell (Heidelberg, Germany). PAECs were cultured in endothelial medium (EBM-2, PromoCell, C-22211) supplemented with 2% fetal calf serum (FCS) and epidermal growth factor (5 ng/ml), basic fibroblast growth factor (10 ng/ml), insulin-like growth factor (20 ng/ml), vascular endothelial growth factor 165 (0.5 ng/ml), ascorbic acid (1 μg/ml), heparin (22.5 μg/ml), and hydrocortisone (0.2 μg/ml) (all from PromoCell). Confluent PAECs from passage 5 through 10 in 2% FCS containing medium were used for all experiments.

We added 100 μL of Matrigel solution (BD Biosciences, 356231) to the wells of 96-well plates and incubated at 37° C for 30 min. 2.0 × 10³ Human umbilical vein endothelial cells (HUVECs, C-12200, PromoCell, Heidelberg, Germany) in 200 μL of endothelial cell basal medium (C-22211, PromoCell) supplemented with growth medium supplement mix (C-39216, PromoCell) and penicillin-streptomycin were seeded on the Matrigel in 96-well plates, and then incubated at 37° C in 5% CO₂ and 1% O₂ for 1 h to have OGD challenge. After OGD, the medium was changed to IM, NSC-CM, or NSC-CM with AG490 (50 μM). After 24 h, the total tube length was measured using the ImageJ software.