Autophagic flux assay using tandem fluorescent-tagged LC3

Human hepatic cells were transfected with GFP-mCherry-LC3 plasmid DNA (Changsha Yingrun Biotechnology Co., Ltd., Changsha, China) according to the manufacturer's protocol. After 24 h, the cells were then treated with the different treatments. The cells were then washed three times with PBS and fixed with 4% paraformaldehyde for 30 min. Images of the cells were captured and observed under a confocal laser scanning microscope (Nikon Corporation, Tokyo, Japan).