2.3. Autophagic flux

LO2 cells were grown on glass chamber slides overnight and then transfected with mCherry‐GFP‐LC3 plasmid for 24 hours. After transfection, cells were treated with rapamycin (1.1 μmol/L), bafilomycin A1 (200 µmol/L), palmitic acid (60 µg/mL) or subject to serum starvation in the absence or presence of polydatin (24 μmol/L). Afterwards, cells were washed twice with PBS and fixed in 4% paraformaldehyde for 15 minutes at room temperature. After rinsing twice with PBS, the slides were mounted in ProLong Gold Anti‐fade reagent (Invitrogen, Carlsbad, CA, USA) and then examined under a confocal microscope (Leica).