Measuring AMPK-Dependent Glucose Uptake

L6 rat myoblasts were cultured overnight at 2 × 10⁴/well in a 96-well plate in DMEM (5 mM glucose) without serum. Cells were washed twice with KRH, pre-treated or not for 30 min at 37 °C with the AMPK inhibitor dorsomorphin (1 µM)^{48 (link)}, and then incubated for 30 min at 37 °C with or without 100 nM ABA. The fluorescently labeled deoxyglucose analog 2-NBDG (50 µM) was added to each well and after 10 min the supernatant was removed, wells were washed once with ice-cold KRH, 50 µL KRH was added to each well and the mean fluorescence (λex 465 nm, λem 540 nm) of 10 acquisitions/well was calculated. Each experimental condition was assayed in at least 8 wells. Unspecific 2-NBDG uptake, determined in the presence of the glucose transport inhibitors cytochalasin B (20 μM) and phloretin (200 μM)^{49 (link)}, was subtracted from each experimental value.