2.6. 2-NBDG Uptake Assay

Glucose uptake activity was analyzed by measuring the uptake of 2-NBDG, a fluorescent D-glucose analogue. C2C12 cells were seeded on 96-well plates (1 × 10³ cells/well) and cultured to 70% confluence in DMEM medium. Then, cells were switched to myoblast differentiation medium containing DMEM supplemented with 2% horse serum, which was replaced every 2 days. Fully differentiated cells were incubated for 24 h in serum-free medium and then treated with 2-NBDG (50 nM) in the presence or absence of MCE, LFE, and MLM at given doses for 24 h. Insulin (100 nM) was used as a positive control for the glucose uptake assay. After washing three times with ice-cold PBS, the intracellular uptake of 2-NBDG was measured using a fluorimeter at excitation and emission wavelengths of 485 and 535 nm, respectively.