Quantification of Glucose Uptake in Adipocytes

Differentiated adipocytes overexpressing or silenced for the expression of both LANCL1 and LANCL2 were cultured overnight at 5 × 10³/well in a 96-well plate in 5 mM DMEM without serum. Cells were washed once with DMEM and then incubated for 5 min at 37 °C in DMEM containing 100 nM ABA. At the end of incubation, cells were washed with KRH at 37 °C. The fluorescently labeled deoxyglucose analog 2-NBDG (50 μM) was added to each well and, after 15 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 (l_ex = 465 nm, l_em = 540 nm) from 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 mM) and phloretin (200 mM) [10 (link)], was subtracted from each experimental value.