2.12. Monitoring autophagic flux by the tandem fluorescent‐tagged LC3 (mCherry‐GFP‐LC3B)

To monitor autophagic flux, cardiomyocytes were transfected with a tandem fluorescent mCherry‐GFP‐tagged LC3 construct as previously reported. 31 The expression of GFP and mCherry was visualized with the confocal scanning microscope (Nikon A1R, Japan). Both GFP and mCherry can be detected in early autophagosomes as yellow puncta. Once the autophagosome and lysosome fuse to form the late autophagolysosome, the green fluorescence quenches and disappears due to the degradation of GFP by the acid lysosomal protease, and the autophagolysosomes were detected as red puncta. Autophagic flux was evaluated by the colour change of GFP/mCherry. Bafilomycin A1 is a vacuolar ATPase inhibitor, an inhibitor of autophagy flux, which prevents the fusion of autophagosomes and lysosomes to form autophagolysosomes. Images were captured at 8h after addition of bafilomycin A1 (100 nmol/L) or DMSO.