Human microvascular retinal endothelial cell line (ACBRI 181, hRECs) was obtained from Cell Systems (Kirkland, WA, USA). As previously described33 , hRECs were grown in fibronectin-coated plate in Dulbecco’s Modified Eagle’s Medium (cat. no. 11885–084) supplemented with 10% fetal calf serum (cat. no. 16000-044) and 100 U/ml of penicillin/streptomycin (cat. no. 15140-122; Gibco; Thermo Fisher Scientific, Inc.). To maintain uniform conditions, all experiments were performed by using passage 4–6 hRECs in this study. Cultured hRECs were also identified by stain of CD13 and von Willebrand Factor (vWF)34 (link); the positive staining of CD13 and vWF has been provided ( Supplemental Fig. 1 ).
hRECs were cultured in the presence of 25 mM of glucose (cat. no. G8270, Sigma-Aldrich) or mannitol (cat. no. PHR1007, Sigma-Aldrich) for different time periods. In some cases, 10 µg/ml bovine serum albumin (BSA; B8667, Sigma-Aldrich), 500 nM PKC inhibitor Go 6976 (cat. no. 2253/1, R&D Systems; Minneapolis, MN), and 10 µM p38 MAPK inhibitor SB203580 (#5633, Cell Signaling Technology) was added to the medium alone or in appropriate combination as indicated.
hRECs were cultured in the presence of 25 mM of glucose (cat. no. G8270, Sigma-Aldrich) or mannitol (cat. no. PHR1007, Sigma-Aldrich) for different time periods. In some cases, 10 µg/ml bovine serum albumin (BSA; B8667, Sigma-Aldrich), 500 nM PKC inhibitor Go 6976 (cat. no. 2253/1, R&D Systems; Minneapolis, MN), and 10 µM p38 MAPK inhibitor SB203580 (#5633, Cell Signaling Technology) was added to the medium alone or in appropriate combination as indicated.