2 n 7 nitrobenz 2 oxa 1 3 diazol 4 yl amino 2 deoxyglucose 2 nbdg

Human RMVECs were plated into 96-well plates at 1 × 10⁴ cells/well and grown for 24 hours. Cells then were grown in serum-free endothelial basal medium (EBM) overnight. After three washes in warm PBS, cells were incubated in Krebs-Ringer-Phosphate-HEPES (KRPH) buffer (20 mM HEPES, 5 mM KH2PO4, 1 mM MgSO4,1 mM CaCl2, 136 mM NaCl, and 4.7 mM KCl, pH 7.4) with 2% BSA for 40 minutes. Cells then were treated with genipin (10 μM) for 2 hours before incubation with VEGF (20 ng/mL). Glucose uptake was measured after incubating with 2-NBDG (2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino]-2-Deoxyglucose, 2-NBDG; 10 μM; Invitrogen) for an additional 30 minutes. DMSO and PBS were used as treatment controls for genipin and VEGF, respectively. After treatment, cells were washed in PBS three times to remove 2-NBDG that was not taken up, and then cells were measured for fluorescence at excitation/emission maxima of approximately 465/540 nm using a fluorescence microplate reader (BioTek, Winooski, VT, USA).

The differentiation and uptake assay was performed as described previously [20 ] with few modifications. Seeded cells were grown overnight and then treated with the methanolic rice cultivar extracts, rosiglitazone (Sigma–Aldrich, USA), apigenin (1:1000) (Sigma–Aldrich, St. Louis, MO, USA) or vehicle control in 100 μL of glucose free culture medium containing 150 μg/ml 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) (Invitrogen Corporation, Carlsbad, CA, USA) and incubated for 90 min. The plates were centrifuged, the supernatant was aspirated and 200 μL of cell based assay buffer was added. The cellular uptake of 2-NBDG was measured using a multifunctional plate reader at excitation and emission wavelengths of 485 and 535 nm, respectively.