2.15. 2-NBDG Glucose Uptake Assay

In accordance with a previous method [6] the glucose uptake assay was established with some modifications. Briefly, HepG2 cells were cultured in 96-well plate to confluence and then treated with 10⁻⁶ M insulin for 24 h to induce insulin resistance (based on a 49% decrease in glucose uptake) (data not shown). Different coumarins (5–40 µM) or 10 µM metformin (non-cytotoxic concentration) concentrations were added and incubated for 24 h, followed by 100 nM insulin and cells incubation for 30 min, respectively. 2-NBDG uptake was measured following this incubation. The cells were incubated with 40 μM of 2-NBDG (dissolved in PBS with 1% BSA) for 20 min. The cells were washed thrice with ice-cold PBS to avoid the reaction, and the fluorescence intensity of 2-NBDG was calculated at 485 nm of excitation and 528 nm of emission on a microplate reader (Bio-Tek Instruments Inc., Winooski, VT, USA). Five replication wells have been set up, and three times each experiment has been replicated.