Measurement of autophagic activity in mammalian cells.

Macroautophagy (referred to herein as autophagy) is a process in which cells engulf portions of cytoplasm in double-membrane vesicles called autophagosomes. These autophagosomes can also capture protein oligomers associated with neurodegenerative diseases, infectious agents (like bacteria), and even organelles (like mitochondria). Autophagosomes are transported along microtubules towards the microtubule organizing center of cells, where the lysosomes are clustered. After fusion and content exchange with lysosomes, the autophagosome cargo is degraded by lysosomal hydrolases. This unit describes some of the core autophagy assays that are in common use, including LC3 immunoblotting, light microscopy analyses of different stages of autophagy, electron microscopy, and an assay of autophagy substrate accumulation. Autophagy assays should generally not be performed alone, but should be accompanied by complementary assays to enable robust interpretations.