Visualizing Autophagy Dynamics with GFP-LC3

Cells were transfected with GFP-LC3 plasmid by using X-treme GENE HP Reagent (Roche, Basel, Switzerland) according to the manufacturer’s instructions. After 48 h transfection, cells were washed with OptiMEM I (Invitrogen, Carlsbad CA, USA, 51985042) and subjected to staining. The cells were stained with LysoTracker^® Green DND probe (Thermo Scientific, Carlsbad, CA, USA, L7526) as recommended by the manufacturer. The formation of GFP-LC3 punctate and Tracker fluorescence were visualized and analyzed using Cytation3 Cell Imaging Multi-Mode Reader (BioTek, Winooski, VT, USA) [20 (link)]. The cells transfected with the GFP-LC3 plasmid were continuously passed to five generations, and the fluorescence expression of the 1st, 3rd, and 5th generation cells was detected to determine the efficiency and stability of transfection (results are not shown).