2.8. Autophagy Flux Assessment by GFP-LC3 Puncta and GFP-LC3 Cleavage Assay

Cells expressing GFP-LC3 were generated by transfection of ARPE-19 cells grown in 6 well-plates and reaching 80% confluency with 2.5 μg pEGFP-LC3 plasmid (Addgene plasmid # 24920) using Lipofectamine 3000 (Invitrogen) for 24 h. After the desired treatments, there was autophagy flux assessment by immunoblotting for free GFP levels in a cell lysate or live-fluorescence cell imaging for GFP-LC3 puncta in the cells. For quantification of GFP-LC3 puncta, treated cells were rinsed with PBS and incubated with serum-free medium for fluorescence imaging using an inverted confocal microscope (Eclipse Ti2-E, Nikon Instruments Europe B.V., Amsterdam) and a 40X objective. GFP-LC3 puncta were quantified from triplicates by counting a total of 30 cells as previously reported [32].