Quantification of Autophagy During Senescence

Abstract

Autophagy constitutes an evolutionarily conserved catabolic process that contributes to the clearance of damaged cellular components in response to a variety of stress conditions. Additionally, it plays a variety of physiological and pathophysiological roles in maintaining cell homeostasis. Recently, the critical role of autophagy during cellular senescence has been supported by evidences demonstrating the reversal of senescence by the reestablishment of autophagy. As considerable attention has been directed toward understanding the molecular mechanisms underlying senescence and autophagy, a method to accurately quantify autophagy during senescence is critical to understand its role in senescence and senescence-related diseases. In this chapter, we describe the use of CYTO-ID® green dye and DQ™ Red BSA to monitor the autophagic flux as an accurate method to quantify autophagic activity. This technique relies on the specificity of CYTO-ID® green dye in staining autophagosome and the cleavage of the self-quenched DQ™ Red BSA protease substrates in an acidic compartment. In particular, herein we describe protocols to quantify autophagy during senescence.