Abstract

Macroautophagy is a major degradation mechanism of cell components via the lysosome. Macroautophagy greatly contributes to not only cell homeostasis but also the prevention of various diseases. Because macroautophagy proceeds through multi‐step reactions, researchers often face a persistent question of how macroautophagic activity can be measured correctly. To make a straightforward determination of macroautophagic activity, diverse monitoring assays have been developed. Direct measurement of lysosome‐dependent degradation of radioisotopically labeled cell proteins has long been applied. Meanwhile, indirect monitoring procedures have been developed. In these assays, autophagosome marker proteins, microtubule‐associated proteins 1A/1B light chain 3B‐II (LC3B‐II) and gamma‐aminobutyric acid receptor‐associated protein‐II (GABARAP‐II) have been analyzed and the validity of the assays strongly depends on appropriate assessment of the fluctuation of LC3‐II and/or GABARAP‐II levels in the presence or absence of lysosomal inhibitors. This article describes these monitoring methods, paying special attention to the principles and characteristics of each procedure.