Methods for Detection of Autophagy in Mammalian Cells

Abstract

Autophagy is a conserved catabolic process that degrades cytoplasmic constituents in the lysosome and thus contributes to the maintenance of intracellular homeostasis. The process of autophagy has been involved in many physiological and pathological processes. Therefore, there is a developing need to identify, quantify, and manipulate the autophagic process accurately in the cells. As autophagy involves dynamic and complex processes, therefore various approaches are needed to study this process precisely. In this chapter, we have tried to elaborate the approaches and methods to monitor autophagy, with a primary focus on mammalian macroautophagy. Autophagy induction can be detected using Western blotting of LC3 (marker protein for autophagosomes) in which LC3-II levels represent the quantity of autophagosomes formed on induction to a particular stimulus. This can also be confirmed by puncta formation assay using confocal microscopy. Further, the autophagic flux can be examined using bafilomycin A1 as inhibitor of autophagosome-lysosome fusion and acidification of lysosomal compartments, thereby leading to accumulation of autophagosomes which is represented by high LC3-II levels. The autophagolysosomal degradation or proteolysis which is the last step of autophagy can be analyzed by DQ-BSA assay.

Keywords: Autophagy; DQ-BSA; LC3; Puncta formation; Western blotting.