Autophagic Flux Detection by mRFP-GFP-LC3 Adenovirus Transfection

mRFP-GFP-LC3 adenovirus (Hanbio Co. Ltd., Shanghai, China) was transfected to HK-2 cells in each confocal cuvette at a titer of 10¹⁰ PFU/mL for 48 h, then the cells were harvested and fixed in 4% paraformaldehyde. The expression of both monomeric red fluorescent protein (mRFP) and green fluorescent protein (GFP) in the mRFP-GFP-LC3 tandem fluorescent proteins were used to track LC3 under a confocal laser microscope. Generally, when autophagosomes form in the cells, LC3 will converge into dots, and the LC3 dots will be tagged with both mRFP, resulting in red fluorescence and GFP, resulting in green fluorescence, simultaneously appearing yellow due to superimposition. However, GFP is sensitive to acidity. When autophagosomes fuse with acidic lysosomes, forming autophagolysosomes, the GFP protein becomes quenched, and the LC3 spots only appear red. Therefore, autophagy in HK-2 cells can be recognized by counting the yellow and red dots. In this study, the numbers of spots of each color were obtained from at least three independent experiments in each group.